t4 dna ligase  (Thermo Fisher)


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    Name:
    T4 DNA Ligase
    Description:
    Thermo Scientific T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex DNA or RNA The enzyme repairs single strand nicks in duplex DNA RNA or DNA RNA hybrids It also joins DNA fragments with either cohesive or blunt termini but has no activity on single stranded nucleic acids T4 DNA Ligase requires ATP as a cofactor Highlights• Active in Themo Scientific restriction enzyme PCR and RT buffers when supplemented with ATP • Fast sticky end ligation is completed in 10 minutes at room temperature• Supplied with PEG solution for efficient blunt end ligationApplications• Cloning of restriction enzyme generated DNA fragments• Cloning of PCR products• Joining of double stranded oligonucleotide linkers or adaptors to DNA• Site directed mutagenesis• Amplified fragment length polymorphism AFLP • Ligase mediated RNA detection see Reference 3 • Nick repair in duplex DNA RNA or DNA RNA hybrids• Self circularization of linear DNA Includes• T4 DNA Ligase• 10X T4 DNA Ligase Buffer• 50 PEG SolutionNotes• Binding of T4 DNA Ligase to DNA may result in a band shift in agarose gels To avoid this incubate samples with 6X DNA Loading Dye SDS Solution at 70°C for 5 min or 65°C for 10 minutes and chill on ice prior to electrophoresis • The volume of the ligation reaction mixture should not exceed 10 of the competent cell volume in the transformation process • Prior to electro transformation remove T4 DNA Ligase from the ligation mixture using spin columns or chloroform extraction The extracted DNA can be further precipitated with ethanol
    Catalog Number:
    el0013
    Price:
    None
    Applications:
    Cloning|Restriction Enzyme Cloning
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher t4 dna ligase
    ( A ) Chemical structures of Ψ and m 6 A. ( B ) Scheme for <t>T4</t> DNA ligase-catalyzed joining of two DNA substrates. In the ternary RNA/DNA complex, the black line corresponds to the 30-mer RNA template with the modified nucleotide (open circle) located at the 15th position. Blue lines correspond to the ligation substrates with the recognition residue shown as a filled blue circle.
    Thermo Scientific T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex DNA or RNA The enzyme repairs single strand nicks in duplex DNA RNA or DNA RNA hybrids It also joins DNA fragments with either cohesive or blunt termini but has no activity on single stranded nucleic acids T4 DNA Ligase requires ATP as a cofactor Highlights• Active in Themo Scientific restriction enzyme PCR and RT buffers when supplemented with ATP • Fast sticky end ligation is completed in 10 minutes at room temperature• Supplied with PEG solution for efficient blunt end ligationApplications• Cloning of restriction enzyme generated DNA fragments• Cloning of PCR products• Joining of double stranded oligonucleotide linkers or adaptors to DNA• Site directed mutagenesis• Amplified fragment length polymorphism AFLP • Ligase mediated RNA detection see Reference 3 • Nick repair in duplex DNA RNA or DNA RNA hybrids• Self circularization of linear DNA Includes• T4 DNA Ligase• 10X T4 DNA Ligase Buffer• 50 PEG SolutionNotes• Binding of T4 DNA Ligase to DNA may result in a band shift in agarose gels To avoid this incubate samples with 6X DNA Loading Dye SDS Solution at 70°C for 5 min or 65°C for 10 minutes and chill on ice prior to electrophoresis • The volume of the ligation reaction mixture should not exceed 10 of the competent cell volume in the transformation process • Prior to electro transformation remove T4 DNA Ligase from the ligation mixture using spin columns or chloroform extraction The extracted DNA can be further precipitated with ethanol
    https://www.bioz.com/result/t4 dna ligase/product/Thermo Fisher
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    t4 dna ligase - by Bioz Stars, 2020-07
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    Related Products / Commonly Used Together

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    t4 dna ligase buffer
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    taq dna polymerase
    dna fragments
    genomic dna
    t4 dna polymerase
    bsa
    bamhi
    mse i
    dna polymerase
    xhoi
    klenow fragment
    dntps

    Images

    1) Product Images from "Identification of recognition residues for ligation-based detection and quantitation of pseudouridine and N6-methyladenosine"

    Article Title: Identification of recognition residues for ligation-based detection and quantitation of pseudouridine and N6-methyladenosine

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm657

    ( A ) Chemical structures of Ψ and m 6 A. ( B ) Scheme for T4 DNA ligase-catalyzed joining of two DNA substrates. In the ternary RNA/DNA complex, the black line corresponds to the 30-mer RNA template with the modified nucleotide (open circle) located at the 15th position. Blue lines correspond to the ligation substrates with the recognition residue shown as a filled blue circle.
    Figure Legend Snippet: ( A ) Chemical structures of Ψ and m 6 A. ( B ) Scheme for T4 DNA ligase-catalyzed joining of two DNA substrates. In the ternary RNA/DNA complex, the black line corresponds to the 30-mer RNA template with the modified nucleotide (open circle) located at the 15th position. Blue lines correspond to the ligation substrates with the recognition residue shown as a filled blue circle.

    Techniques Used: Modification, Ligation

    2) Product Images from "Practical Synthesis of Cap‐4 RNA"

    Article Title: Practical Synthesis of Cap‐4 RNA

    Journal: Chembiochem

    doi: 10.1002/cbic.201900590

    Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using T4 DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μ m RNA 10 , 12.5 μ m RNA 11 , 12.5 μ m splint; 0.5 m m ATP, 40 m m Tris ⋅ HCl (pH 7.8), 10 m m MgCl 2 , 10 m m DTT, 5 % ( w / v ) PEG 4000, 0.5 U μL −1 T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.
    Figure Legend Snippet: Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using T4 DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μ m RNA 10 , 12.5 μ m RNA 11 , 12.5 μ m splint; 0.5 m m ATP, 40 m m Tris ⋅ HCl (pH 7.8), 10 m m MgCl 2 , 10 m m DTT, 5 % ( w / v ) PEG 4000, 0.5 U μL −1 T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.

    Techniques Used: Ligation, Sequencing, High Performance Liquid Chromatography, Purification

    3) Product Images from "Profiling non-lysyl tRNAs in HIV-1"

    Article Title: Profiling non-lysyl tRNAs in HIV-1

    Journal: RNA

    doi: 10.1261/rna.1928110

    Detection of tRNAs by microarray analysis. ( A ) Array scheme. Total cellular or viral RNA were deacylated, and directly labeled with a Cy3 or Cy5 containing oligonucleotide using T4 DNA ligase. The labeling samples with the opposite fluorophores were combined
    Figure Legend Snippet: Detection of tRNAs by microarray analysis. ( A ) Array scheme. Total cellular or viral RNA were deacylated, and directly labeled with a Cy3 or Cy5 containing oligonucleotide using T4 DNA ligase. The labeling samples with the opposite fluorophores were combined

    Techniques Used: Microarray, Labeling

    4) Product Images from "Autophosphorylation-dependent remodeling of the DNA-dependent protein kinase catalytic subunit regulates ligation of DNA ends"

    Article Title: Autophosphorylation-dependent remodeling of the DNA-dependent protein kinase catalytic subunit regulates ligation of DNA ends

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh761

    DNA-PKcs kinase activity regulates T4 DNA ligase-mediated DNA end joining. Purified DNA-PKcs (1 μg, lanes 10–15) and/or Ku (0.5 μg, lanes 6–8 and 13–15) were incubated in the absence (lanes 5 and 9) or presence (lanes 6–8 and 10–15) of T4-DNA ligase. Lanes 2–4 contained T4 DNA ligase alone (no Ku or DNA-PKcs). Lane 1 contained linearized ds plasmid DNA alone. Samples in lanes 4, 8, 12 and 15 were pre-incubated with wortmannin (W) (1 μM) or an equivalent volume of DMSO (D) (lanes 3, 7, 11 and 14). Reactions were stopped after 30 min and analyzed as described in Materials and Methods.
    Figure Legend Snippet: DNA-PKcs kinase activity regulates T4 DNA ligase-mediated DNA end joining. Purified DNA-PKcs (1 μg, lanes 10–15) and/or Ku (0.5 μg, lanes 6–8 and 13–15) were incubated in the absence (lanes 5 and 9) or presence (lanes 6–8 and 10–15) of T4-DNA ligase. Lanes 2–4 contained T4 DNA ligase alone (no Ku or DNA-PKcs). Lane 1 contained linearized ds plasmid DNA alone. Samples in lanes 4, 8, 12 and 15 were pre-incubated with wortmannin (W) (1 μM) or an equivalent volume of DMSO (D) (lanes 3, 7, 11 and 14). Reactions were stopped after 30 min and analyzed as described in Materials and Methods.

    Techniques Used: Activity Assay, Purification, Incubation, Plasmid Preparation

    Model for the role of DNA-PKcs autophosphorylation in DNA end ligation. In the first step on NHEJ, Ku (indicated by the small, closed circles) binds to the ends of the DSB. DNA-PKcs (large gray ellipses) is recruited to form the active DNA-PK complex. Upon synapsis, DNA-PK undergoes autophosphorylation, which causes a conformational change that renders the DNA ends accessible to end joining by T4 DNA ligase or the XRCC4/DNA-ligase IV complex.
    Figure Legend Snippet: Model for the role of DNA-PKcs autophosphorylation in DNA end ligation. In the first step on NHEJ, Ku (indicated by the small, closed circles) binds to the ends of the DSB. DNA-PKcs (large gray ellipses) is recruited to form the active DNA-PK complex. Upon synapsis, DNA-PK undergoes autophosphorylation, which causes a conformational change that renders the DNA ends accessible to end joining by T4 DNA ligase or the XRCC4/DNA-ligase IV complex.

    Techniques Used: Ligation, Non-Homologous End Joining

    5) Product Images from "Actual Ligation Frequencies in the Chromosome Conformation Capture Procedure"

    Article Title: Actual Ligation Frequencies in the Chromosome Conformation Capture Procedure

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0060403

    The kinetics of the ligation reaction in the presence or absence of SDS. Electrophoretic separation of the products obtained upon the ligation of pUC18 Hind III fragments for the indicated times in the presence or absence of SDS and Triton X-100 in an ethidium bromide-stained agarose gel. The reaction was carried out in 1× T4 DNA Ligase Buffer (Fermentas) with 100 ng/ µl DNA and 0.1 U/ µl T4 DNA ligase (Fermentas). M–DNA size marker (Fermentas, SM0331).
    Figure Legend Snippet: The kinetics of the ligation reaction in the presence or absence of SDS. Electrophoretic separation of the products obtained upon the ligation of pUC18 Hind III fragments for the indicated times in the presence or absence of SDS and Triton X-100 in an ethidium bromide-stained agarose gel. The reaction was carried out in 1× T4 DNA Ligase Buffer (Fermentas) with 100 ng/ µl DNA and 0.1 U/ µl T4 DNA ligase (Fermentas). M–DNA size marker (Fermentas, SM0331).

    Techniques Used: Ligation, Staining, Agarose Gel Electrophoresis, Marker

    6) Product Images from "FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis"

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2019.4355

    Construction of the knockdown vector pCB309-PFUFT. The FSH1 cDNA was ligated into pUC-PUT after DNA digestion by Xho I and Hin dIII to construct plasmid pUC-PFUFT. The two plasmids, pUC-PFUFT and pCB309 were digested by Spe I and Sac I and ligated with T4 DNA ligase to construct the final FSH1 double stranded RNA interference plasmid pCB309-PFUFT. FSH1, family of serine hydrolases 1.
    Figure Legend Snippet: Construction of the knockdown vector pCB309-PFUFT. The FSH1 cDNA was ligated into pUC-PUT after DNA digestion by Xho I and Hin dIII to construct plasmid pUC-PFUFT. The two plasmids, pUC-PFUFT and pCB309 were digested by Spe I and Sac I and ligated with T4 DNA ligase to construct the final FSH1 double stranded RNA interference plasmid pCB309-PFUFT. FSH1, family of serine hydrolases 1.

    Techniques Used: Plasmid Preparation, Construct

    7) Product Images from "Reverse transcriptase and endonuclease activities encoded by Penelope-like retroelements"

    Article Title: Reverse transcriptase and endonuclease activities encoded by Penelope-like retroelements

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0406281101

    Properties of Penelope -encoded EN and its mutant derivatives. ( A and B ) Cleavage and DNA-binding assays. The 38-bp ps1 target ( A Upper ) was labeled with [α- 32 P]dATP at both ends (in bold italics). Lane C, control without protein. The remaining seven lanes in A and B represent cleavage and DNA-binding assays, respectively, for the EN protein and its mutant variants: Y730H (2%); Y746H (0%); R757A (100%); H781G (27%); D792A (5%); E800G (0%); and WT, wild-type Penelope EN (100%); numbers in parentheses designate the relative cleavage activity with respect to WT obtained from PhosphorImager quantitative scans of gel A . To suppress target cleavage, binding reactions in B were performed without Mg 2+ and in the presence of EDTA; however, residual cleavage is still visible for the most active R757A and WT proteins. ( C ) Binding of WT Penelope EN to the ps1 target at different concentrations of poly(dI)·poly(dC). Lane C, control without protein; WT, wild-type Penelope EN in a standard binding reaction with 0.1 mg/ml BSA. In the remaining lanes, increasing amounts of poly(dI)·poly(dC) (from 0.025 to 1 μg) were added to the binding reaction, as indicated. ( D ) Determination of cleavage sites of Penelope EN. The 34-bp fragment of ps1 was used as a target, with the top and bottom strand labeled as indicated. Lane L, 1-bp DNA ladder; WT, labeled fragments digested by WT EN; Y746H, labeled fragments digested by the EN mutant. Target site duplication (TSD) is shown by brackets, and the most prominent cleavage products, which become visible after a 1-h incubation, are shown in bold italics. ( E ) Ligation of the labeled ps1 fragment after digestion by the WT Penelope EN. Lane C, labeled fragment without protein; WT, labeled fragment digested with EN. The remaining lanes show ligation of the cleavage products after 3-, 10-, and 30-min incubation with T4 DNA ligase.
    Figure Legend Snippet: Properties of Penelope -encoded EN and its mutant derivatives. ( A and B ) Cleavage and DNA-binding assays. The 38-bp ps1 target ( A Upper ) was labeled with [α- 32 P]dATP at both ends (in bold italics). Lane C, control without protein. The remaining seven lanes in A and B represent cleavage and DNA-binding assays, respectively, for the EN protein and its mutant variants: Y730H (2%); Y746H (0%); R757A (100%); H781G (27%); D792A (5%); E800G (0%); and WT, wild-type Penelope EN (100%); numbers in parentheses designate the relative cleavage activity with respect to WT obtained from PhosphorImager quantitative scans of gel A . To suppress target cleavage, binding reactions in B were performed without Mg 2+ and in the presence of EDTA; however, residual cleavage is still visible for the most active R757A and WT proteins. ( C ) Binding of WT Penelope EN to the ps1 target at different concentrations of poly(dI)·poly(dC). Lane C, control without protein; WT, wild-type Penelope EN in a standard binding reaction with 0.1 mg/ml BSA. In the remaining lanes, increasing amounts of poly(dI)·poly(dC) (from 0.025 to 1 μg) were added to the binding reaction, as indicated. ( D ) Determination of cleavage sites of Penelope EN. The 34-bp fragment of ps1 was used as a target, with the top and bottom strand labeled as indicated. Lane L, 1-bp DNA ladder; WT, labeled fragments digested by WT EN; Y746H, labeled fragments digested by the EN mutant. Target site duplication (TSD) is shown by brackets, and the most prominent cleavage products, which become visible after a 1-h incubation, are shown in bold italics. ( E ) Ligation of the labeled ps1 fragment after digestion by the WT Penelope EN. Lane C, labeled fragment without protein; WT, labeled fragment digested with EN. The remaining lanes show ligation of the cleavage products after 3-, 10-, and 30-min incubation with T4 DNA ligase.

    Techniques Used: Mutagenesis, Binding Assay, Labeling, Activity Assay, Incubation, Ligation

    8) Product Images from "The Δ133p53 Isoform Reduces Wtp53-induced Stimulation of DNA Pol γ Activity in the Presence and Absence of D4T"

    Article Title: The Δ133p53 Isoform Reduces Wtp53-induced Stimulation of DNA Pol γ Activity in the Presence and Absence of D4T

    Journal: Aging and Disease

    doi: 10.14336/AD.2016.0910

    In vitro BER assay with purified wtP53, Δ40p53 and Δ133p53 fusion proteins showing that Δ40p53 and Δ133p53 cannot induce mtBER but can attenuate mtBER activity induced by wtp53 . (A) wtP53, Δ40p53 and Δ133p53 His fusion proteins were stained with Coomassie blue (upper panel) and identified by Western blotting with anti-P53 antibodies (lower panel). (B) Purified p53, Δ40p53 and Δ133p53 protein (100, 500 and 1000 ng, lanes 3-9) or d4T (10, 50 and 300 nM, lanes 11-14) were added to BER reaction mixtures containing both whole-mitochondrial extracts obtained from H1299 cells and T4 DNA ligase. The templates were treated with T4 DNA ligase and Klenow fragment was used as a positive control (lane 15).
    Figure Legend Snippet: In vitro BER assay with purified wtP53, Δ40p53 and Δ133p53 fusion proteins showing that Δ40p53 and Δ133p53 cannot induce mtBER but can attenuate mtBER activity induced by wtp53 . (A) wtP53, Δ40p53 and Δ133p53 His fusion proteins were stained with Coomassie blue (upper panel) and identified by Western blotting with anti-P53 antibodies (lower panel). (B) Purified p53, Δ40p53 and Δ133p53 protein (100, 500 and 1000 ng, lanes 3-9) or d4T (10, 50 and 300 nM, lanes 11-14) were added to BER reaction mixtures containing both whole-mitochondrial extracts obtained from H1299 cells and T4 DNA ligase. The templates were treated with T4 DNA ligase and Klenow fragment was used as a positive control (lane 15).

    Techniques Used: In Vitro, Purification, Activity Assay, Staining, Western Blot, Positive Control

    9) Product Images from "Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain"

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039251

    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.
    Figure Legend Snippet: 15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    12% denaturing PAGE for the ligation products of linkers A–B treated with CIAP. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.
    Figure Legend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B treated with CIAP. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Positive Control, Negative Control

    12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.
    Figure Legend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.
    Figure Legend Snippet: The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Electrophoresis, Negative Control, Positive Control

    10) Product Images from "In vitro repair of complex unligatable oxidatively induced DNA double-strand breaks by human cell extracts"

    Article Title: In vitro repair of complex unligatable oxidatively induced DNA double-strand breaks by human cell extracts

    Journal: Nucleic Acids Research

    doi:

    Differential repair of bleomycin-induced DSB ends in comparison with Stu I-induced blunt ends. Standard repair reactions were performed with either 100 ng substrate DNA linearized by 0.5 µg bleomycin/ml or Stu I digestion. The repair reactions contained either 15 µg PTN A, 2 U E.coli endonuclease IV (Endo IV), 5 U T4 DNA ligase (T4 Ligase) or combinations as indicated. Reactions were incubated at 17°C for 18 h. ( A ) The reactions were as follows: lane 1, bleomycin (blm)-damaged DNA negative control; lane 2, blm-damaged DNA + PTN A; lane 3, blm-damaged DNA + endo IV; lane 4, blm-damaged DNA + T4 ligase; lane 5, blm-damaged DNA + endo IV + T4 ligase; lane 6, Stu I cut (blunt-end) DNA negative control; lane 7, Stu I cut DNA + PTN A; lane 8, Stu I cut DNA + endo IV; lane 9, Stu I cut DNA + T4 ligase; lane 10, Stu I cut DNA + endo IV + T4 ligase. DNA substrate and product forms are indicated as linear (L), circular (C), dimer (D), trimer (T) and high molecular weight multimers (HM) to the right of the gel. ( B ) The data in the gel were plotted as a percentage of linear substrate DNA converted to rejoined products. Reactions performed with bleomycin-linearized DNA substrates are indicated by gray bars. Reactions conducted with the Stu I-linearized blunt-end DSB substrates are indicated by black bars.
    Figure Legend Snippet: Differential repair of bleomycin-induced DSB ends in comparison with Stu I-induced blunt ends. Standard repair reactions were performed with either 100 ng substrate DNA linearized by 0.5 µg bleomycin/ml or Stu I digestion. The repair reactions contained either 15 µg PTN A, 2 U E.coli endonuclease IV (Endo IV), 5 U T4 DNA ligase (T4 Ligase) or combinations as indicated. Reactions were incubated at 17°C for 18 h. ( A ) The reactions were as follows: lane 1, bleomycin (blm)-damaged DNA negative control; lane 2, blm-damaged DNA + PTN A; lane 3, blm-damaged DNA + endo IV; lane 4, blm-damaged DNA + T4 ligase; lane 5, blm-damaged DNA + endo IV + T4 ligase; lane 6, Stu I cut (blunt-end) DNA negative control; lane 7, Stu I cut DNA + PTN A; lane 8, Stu I cut DNA + endo IV; lane 9, Stu I cut DNA + T4 ligase; lane 10, Stu I cut DNA + endo IV + T4 ligase. DNA substrate and product forms are indicated as linear (L), circular (C), dimer (D), trimer (T) and high molecular weight multimers (HM) to the right of the gel. ( B ) The data in the gel were plotted as a percentage of linear substrate DNA converted to rejoined products. Reactions performed with bleomycin-linearized DNA substrates are indicated by gray bars. Reactions conducted with the Stu I-linearized blunt-end DSB substrates are indicated by black bars.

    Techniques Used: Incubation, Negative Control, Molecular Weight

    11) Product Images from "Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase"

    Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx033

    Single-stranded DNA ligation with T4 DNA ligase and CircLigase. A pool of 60 nt acceptor oligonucleotides (‘60N’) were ligated to 10 pmol of a 3΄ biotinylated donor oligonucleotide (CL78) using either T4 DNA ligase in the presence of a splinter oligonucleotide (TL38) or CircLigase. Ligation products were visualized on a 10% denaturing polyacrylamide gel stained with SybrGold. Band shifts from 60 nt to 80 nt indicate successful ligation. Schematic overviews of the reaction schemes are shown on top. The scheme developed by Kwok et al . ( 19 ) is shown for comparison. M: Single-stranded DNA size marker.
    Figure Legend Snippet: Single-stranded DNA ligation with T4 DNA ligase and CircLigase. A pool of 60 nt acceptor oligonucleotides (‘60N’) were ligated to 10 pmol of a 3΄ biotinylated donor oligonucleotide (CL78) using either T4 DNA ligase in the presence of a splinter oligonucleotide (TL38) or CircLigase. Ligation products were visualized on a 10% denaturing polyacrylamide gel stained with SybrGold. Band shifts from 60 nt to 80 nt indicate successful ligation. Schematic overviews of the reaction schemes are shown on top. The scheme developed by Kwok et al . ( 19 ) is shown for comparison. M: Single-stranded DNA size marker.

    Techniques Used: DNA Ligation, Ligation, Staining, Marker

    Library preparation methods for highly degraded DNA. ( A ) In the single-stranded library preparation method described here (ssDNA2.0), DNA fragments (black) are 5΄ and 3΄ dephosphorylated and separated into single strands by heat denaturation. 3΄ biotinylated adapter molecules (red) are attached to the 3΄ ends of the DNA fragments via hybridization to a stretch of six random nucleotides (marked as ‘N’) belonging to a splinter oligonucleotide complementary to the adapter and nick closure with T4 DNA ligase. Following the immobilization of the ligation products on streptavidin-coated beads, the splinter oligonucleotide is removed by bead wash at an elevated temperature. Synthesis of the second strand is carried out using the Klenow fragment of Escherichia coli DNA polymerase I and a primer with phosphorothioate backbone modifications (red stars) to prevent exonucleolytic degradation. Unincorporated primers are removed through a bead wash at an elevated temperature, preventing the formation of adapter dimers in the subsequent blunt-end ligation reaction, which is again catalyzed by T4 DNA ligase. Adapter self-ligation is prevented through a 3΄ dideoxy modification in the adapter. The final library strand is released from the beads by heat denaturation. ( B ) In the single-stranded library preparation method originally described in Gansauge and Meyer, ( 4 ), the first adapter was attached through true single-stranded DNA ligation using CircLigase. The large fragment of Bst DNA polymerase was used to copy the template strand, leaving overhanging 3΄ nucleotides, which had to be removed in a blunt-end repair reaction using T4 DNA polymerase. ( C ) The ‘454’ method of double-stranded library preparation in the implementation of Meyer and Kircher, ( 23 ), is based on non-directional blunt-end ligation of a mixture of two adapters to blunt-end repaired DNA fragments using T4 DNA ligase. To prevent adapter self-ligation, no phosphate groups are present at the 5΄ ends of the adapters, resulting in the ligation of the adapter strands only and necessitating subsequent nick fill-in with a strand-displacing polymerase. Intermittent DNA purification steps are required in-between enzymatic reactions. ( D ) The ‘Illumina’ method of double-stranded library preparation, shown here as implemented in New England Biolabs’ NEBNext Ultra II kit, requires the addition of A-overhangs (marked as ‘A’) to blunt-end repaired DNA fragments using a 3΄-5΄ exonuclease deletion mutant of the Klenow fragment of E. coli DNA polymerase I. Both adapter sequences are combined into one bell-shaped structure, which carries a 3΄ T overhang to allow sticky end ligation with T4 DNA ligase. Following ligation, adapter strands are separated by excision of uracil. Excess adapters and adapter dimers are removed through size-selective purification.
    Figure Legend Snippet: Library preparation methods for highly degraded DNA. ( A ) In the single-stranded library preparation method described here (ssDNA2.0), DNA fragments (black) are 5΄ and 3΄ dephosphorylated and separated into single strands by heat denaturation. 3΄ biotinylated adapter molecules (red) are attached to the 3΄ ends of the DNA fragments via hybridization to a stretch of six random nucleotides (marked as ‘N’) belonging to a splinter oligonucleotide complementary to the adapter and nick closure with T4 DNA ligase. Following the immobilization of the ligation products on streptavidin-coated beads, the splinter oligonucleotide is removed by bead wash at an elevated temperature. Synthesis of the second strand is carried out using the Klenow fragment of Escherichia coli DNA polymerase I and a primer with phosphorothioate backbone modifications (red stars) to prevent exonucleolytic degradation. Unincorporated primers are removed through a bead wash at an elevated temperature, preventing the formation of adapter dimers in the subsequent blunt-end ligation reaction, which is again catalyzed by T4 DNA ligase. Adapter self-ligation is prevented through a 3΄ dideoxy modification in the adapter. The final library strand is released from the beads by heat denaturation. ( B ) In the single-stranded library preparation method originally described in Gansauge and Meyer, ( 4 ), the first adapter was attached through true single-stranded DNA ligation using CircLigase. The large fragment of Bst DNA polymerase was used to copy the template strand, leaving overhanging 3΄ nucleotides, which had to be removed in a blunt-end repair reaction using T4 DNA polymerase. ( C ) The ‘454’ method of double-stranded library preparation in the implementation of Meyer and Kircher, ( 23 ), is based on non-directional blunt-end ligation of a mixture of two adapters to blunt-end repaired DNA fragments using T4 DNA ligase. To prevent adapter self-ligation, no phosphate groups are present at the 5΄ ends of the adapters, resulting in the ligation of the adapter strands only and necessitating subsequent nick fill-in with a strand-displacing polymerase. Intermittent DNA purification steps are required in-between enzymatic reactions. ( D ) The ‘Illumina’ method of double-stranded library preparation, shown here as implemented in New England Biolabs’ NEBNext Ultra II kit, requires the addition of A-overhangs (marked as ‘A’) to blunt-end repaired DNA fragments using a 3΄-5΄ exonuclease deletion mutant of the Klenow fragment of E. coli DNA polymerase I. Both adapter sequences are combined into one bell-shaped structure, which carries a 3΄ T overhang to allow sticky end ligation with T4 DNA ligase. Following ligation, adapter strands are separated by excision of uracil. Excess adapters and adapter dimers are removed through size-selective purification.

    Techniques Used: Hybridization, Ligation, Modification, DNA Ligation, DNA Purification, Mutagenesis, Purification

    Effects of single-stranded ligation schemes on library characteristics. ( A ) Informative sequence content of libraries prepared with CircLigase and T4 DNA ligase as a function of the input volume of ancient DNA extract used for library preparation. ( B ) Average GC content of the sequences obtained with the two ligation schemes. Note that the average GC content exceeds that of a typical mammalian genome because most sequences derive from microbial DNA, which is the dominant source of DNA in most ancient bones. ( C ) Fragment size distribution in the libraries as inferred from overlap-merged paired-end reads. Short artifacts in the library prepared from extremely little input DNA (corresponding to ∼1 mg bone) are mainly due to the incorporation of splinter fragments. ( D ) Frequencies of damage-induced C to T substitutions near the 5΄ and 3΄ ends of sequences.
    Figure Legend Snippet: Effects of single-stranded ligation schemes on library characteristics. ( A ) Informative sequence content of libraries prepared with CircLigase and T4 DNA ligase as a function of the input volume of ancient DNA extract used for library preparation. ( B ) Average GC content of the sequences obtained with the two ligation schemes. Note that the average GC content exceeds that of a typical mammalian genome because most sequences derive from microbial DNA, which is the dominant source of DNA in most ancient bones. ( C ) Fragment size distribution in the libraries as inferred from overlap-merged paired-end reads. Short artifacts in the library prepared from extremely little input DNA (corresponding to ∼1 mg bone) are mainly due to the incorporation of splinter fragments. ( D ) Frequencies of damage-induced C to T substitutions near the 5΄ and 3΄ ends of sequences.

    Techniques Used: Ligation, Sequencing, Ancient DNA Assay

    12) Product Images from "FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis"

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2019.4355

    Construction of the knockdown vector pCB309-PFUFT. The FSH1 cDNA was ligated into pUC-PUT after DNA digestion by Xho I and Hin dIII to construct plasmid pUC-PFUFT. The two plasmids, pUC-PFUFT and pCB309 were digested by Spe I and Sac I and ligated with T4 DNA ligase to construct the final FSH1 double stranded RNA interference plasmid pCB309-PFUFT. FSH1, family of serine hydrolases 1.
    Figure Legend Snippet: Construction of the knockdown vector pCB309-PFUFT. The FSH1 cDNA was ligated into pUC-PUT after DNA digestion by Xho I and Hin dIII to construct plasmid pUC-PFUFT. The two plasmids, pUC-PFUFT and pCB309 were digested by Spe I and Sac I and ligated with T4 DNA ligase to construct the final FSH1 double stranded RNA interference plasmid pCB309-PFUFT. FSH1, family of serine hydrolases 1.

    Techniques Used: Plasmid Preparation, Construct

    13) Product Images from "Extract of Nippostrongylus brasiliensis Stimulates Polyclonal Type-2 Immunoglobulin Response by Inducing De Novo Class Switch"

    Article Title: Extract of Nippostrongylus brasiliensis Stimulates Polyclonal Type-2 Immunoglobulin Response by Inducing De Novo Class Switch

    Journal: Infection and Immunity

    doi:

    AWH-induced IgG1 production is associated with an increase in the number of IgG1-switched cells. Genomic DNA was isolated from either the TSI-18 and IB4 hybridomas (A) or the spleen cells of mice treated with either AWH, worms ( Nb ), or FIA (B) as described in Materials and Methods. The DNA was digested with Eco RI, ligated with T4 DNA ligase, and amplified by PCR using primers specific for the recombined switch regions. nAChRe levels in all samples were also determined by DC-PCR to control for equal template loading and allow semiquantitation (comparison) of the Sμ-Sγ1 product. PCR amplicons were resolved on a 1.5% agarose gel with ethidium bromide staining. Results are representative of six experiments. (A) Lane 1, TSI-18 (IgG1-producing hybridoma); lane 2, IB4 (IgG2a-producing hybridoma); lane 3, no DNA (control for PCR contamination); lane 4, TSI-18 (nAChRe amplicon from IgG1-producing hybridoma).
    Figure Legend Snippet: AWH-induced IgG1 production is associated with an increase in the number of IgG1-switched cells. Genomic DNA was isolated from either the TSI-18 and IB4 hybridomas (A) or the spleen cells of mice treated with either AWH, worms ( Nb ), or FIA (B) as described in Materials and Methods. The DNA was digested with Eco RI, ligated with T4 DNA ligase, and amplified by PCR using primers specific for the recombined switch regions. nAChRe levels in all samples were also determined by DC-PCR to control for equal template loading and allow semiquantitation (comparison) of the Sμ-Sγ1 product. PCR amplicons were resolved on a 1.5% agarose gel with ethidium bromide staining. Results are representative of six experiments. (A) Lane 1, TSI-18 (IgG1-producing hybridoma); lane 2, IB4 (IgG2a-producing hybridoma); lane 3, no DNA (control for PCR contamination); lane 4, TSI-18 (nAChRe amplicon from IgG1-producing hybridoma).

    Techniques Used: Isolation, Mouse Assay, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    14) Product Images from "Multiplex Single-Molecule DNA Barcoding Using an Oligonucleotide Ligation Assay"

    Article Title: Multiplex Single-Molecule DNA Barcoding Using an Oligonucleotide Ligation Assay

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2018.08.013

    DNA barcoding experimental scheme. Target DNA strands are immobilized on a microscope slide, and dye-labeled barcodes are introduced together with T4 DNA ligase in the microfluidic chamber (1). Complementary barcodes bind transiently to the target site (2), whereas mismatched barcodes bind on an even shorter timescale (2′). Successful ligation is observed for the complementary barcodes (3) but not for the mismatched barcodes (3′). Ligation product shows stable binding to the target DNA (4), whereas mismatched barcodes dissociate and are washed away before imaging. To see this figure in color, go online.
    Figure Legend Snippet: DNA barcoding experimental scheme. Target DNA strands are immobilized on a microscope slide, and dye-labeled barcodes are introduced together with T4 DNA ligase in the microfluidic chamber (1). Complementary barcodes bind transiently to the target site (2), whereas mismatched barcodes bind on an even shorter timescale (2′). Successful ligation is observed for the complementary barcodes (3) but not for the mismatched barcodes (3′). Ligation product shows stable binding to the target DNA (4), whereas mismatched barcodes dissociate and are washed away before imaging. To see this figure in color, go online.

    Techniques Used: Microscopy, Labeling, Ligation, Binding Assay, Imaging

    15) Product Images from "Increasing the efficiency of SAGE adaptor ligation by directed ligation chemistry"

    Article Title: Increasing the efficiency of SAGE adaptor ligation by directed ligation chemistry

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnh082

    Ligation of SAGE adaptor 1A to anchored 3′ end cDNA. An aliquot of 100 ng of in vitro transcribed polyadenylated product was processed under the microSAGE protocol and split into half. Lane 2 shows a control reaction in which T4 DNA ligase was not added to the ligation mixture. Lane 3 shows the formation of a small amount of the hetero-ligation product indicated by the arrow as well as a high molecular weight band corresponding to twice the molecular weight of the unligated cDNA. Ligations were performed as described in Materials and Methods.
    Figure Legend Snippet: Ligation of SAGE adaptor 1A to anchored 3′ end cDNA. An aliquot of 100 ng of in vitro transcribed polyadenylated product was processed under the microSAGE protocol and split into half. Lane 2 shows a control reaction in which T4 DNA ligase was not added to the ligation mixture. Lane 3 shows the formation of a small amount of the hetero-ligation product indicated by the arrow as well as a high molecular weight band corresponding to twice the molecular weight of the unligated cDNA. Ligations were performed as described in Materials and Methods.

    Techniques Used: Ligation, In Vitro, Molecular Weight

    16) Product Images from "Biochemical and structural characterization of Cren7, a novel chromatin protein conserved among Crenarchaea"

    Article Title: Biochemical and structural characterization of Cren7, a novel chromatin protein conserved among Crenarchaea

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm1128

    Interaction of Cren7 with DNA in vitro . ( A ) Comparison of binding of Cren7 to dsDNA and ssDNA. Recombinant Cren7 was incubated with a radiolabeled 60 bp dsDNA or 60 nt ssDNA. Protein–DNA complexes were subjected to electrophoresis in polyacrylamide. Protein concentrations were 0, 0.02, 0.04, 0.08, 0.16, 0.31, 0.63, 1.3, 2.5, 5 and 10 μM, respectively. ( B ) Effect of binding by Cren7 on thermal stability of dsDNA. Thermal denaturation of poly[dAdT]–poly[dAdT] in the presence and absence of Cren7 was determined by monitoring changes in UV absorbance at 260 nm. ( C ) Nick closure analysis of the ability of Cren7 to constrain DNA supercoils. Single-nicked plasmid pBR322 was incubated with Cren7 at various protein/DNA mass ratios and ligated with T4 DNA ligase at 25°C. Samples were deproteinized and subjected to agarose gel electrophoresis. Lane 1, negatively supercoiled pBR322; lane 2, single-nicked pBR322; lanes 3–10, topoisomers of pBR322 ligated in the presence of Cren7 at the following protein/DNA mass ratios: 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.375 and 0.5, respectively. N, nicked circular plasmid; L, linear plasmid; S, supercoiled plasmid. ( D ) Plot of the linking number change of nick-closed plasmid against the Cren7/DNA mass ratio. The linking change of pBR322 ligated in the presence or absence of Cren7 was measured by resolving topoisomers on agarose gels in the presence or absence of chloroquine and band counting.
    Figure Legend Snippet: Interaction of Cren7 with DNA in vitro . ( A ) Comparison of binding of Cren7 to dsDNA and ssDNA. Recombinant Cren7 was incubated with a radiolabeled 60 bp dsDNA or 60 nt ssDNA. Protein–DNA complexes were subjected to electrophoresis in polyacrylamide. Protein concentrations were 0, 0.02, 0.04, 0.08, 0.16, 0.31, 0.63, 1.3, 2.5, 5 and 10 μM, respectively. ( B ) Effect of binding by Cren7 on thermal stability of dsDNA. Thermal denaturation of poly[dAdT]–poly[dAdT] in the presence and absence of Cren7 was determined by monitoring changes in UV absorbance at 260 nm. ( C ) Nick closure analysis of the ability of Cren7 to constrain DNA supercoils. Single-nicked plasmid pBR322 was incubated with Cren7 at various protein/DNA mass ratios and ligated with T4 DNA ligase at 25°C. Samples were deproteinized and subjected to agarose gel electrophoresis. Lane 1, negatively supercoiled pBR322; lane 2, single-nicked pBR322; lanes 3–10, topoisomers of pBR322 ligated in the presence of Cren7 at the following protein/DNA mass ratios: 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.375 and 0.5, respectively. N, nicked circular plasmid; L, linear plasmid; S, supercoiled plasmid. ( D ) Plot of the linking number change of nick-closed plasmid against the Cren7/DNA mass ratio. The linking change of pBR322 ligated in the presence or absence of Cren7 was measured by resolving topoisomers on agarose gels in the presence or absence of chloroquine and band counting.

    Techniques Used: In Vitro, Binding Assay, Recombinant, Incubation, Electrophoresis, Plasmid Preparation, Agarose Gel Electrophoresis

    17) Product Images from "IRDL Cloning: A One-Tube, Zero-Background, Easy-to-Use, Directional Cloning Method Improves Throughput in Recombinant DNA Preparation"

    Article Title: IRDL Cloning: A One-Tube, Zero-Background, Easy-to-Use, Directional Cloning Method Improves Throughput in Recombinant DNA Preparation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0107907

    Schematic diagram of cloning the gene of interest containing internal restriction sites into expression vector. A, generation of sticky-end fragments and cloning into pWXY1.0 by IRDL cloning. The JcDGAT2 was amplified by two pairs of primers, P1–P2, and P3–P4, which were appended with short sequence tails: C, TCGAG, AATTC, G, respectively. After gel-purification, the two PCR products were mixed together, denatured, and reannealed, resulting in 25% of the DNA fragments with Eco RI and XhoI overhangs. Concomitantly, the vector was double-digested with EcoRI and XhoI for 5–10 min at 37°C. After heat inactivation of the restriction enzyme at 95°C for 5 min, the vector was mixed with the reannealed JcDGAT2 containing EcoRI and XhoI overhangs, T4 DNA ligase and ATP were added and incubated at room temperature for 20 min, and finally transformed into E. coli . B, Restriction digestion (EcoRI and XhoI) of minipreps of pWXY1.0 (V) and pWXY1.0-JcDGAT2 (lane 1 to lane 10). The restriction pattern of pWXY1.0-JcDGAT2 generated by EcoRI and XhoI digestion was as predicted: 861 bp and 200 bp, respectively. M, DNA ruler DL2502 (Generay).
    Figure Legend Snippet: Schematic diagram of cloning the gene of interest containing internal restriction sites into expression vector. A, generation of sticky-end fragments and cloning into pWXY1.0 by IRDL cloning. The JcDGAT2 was amplified by two pairs of primers, P1–P2, and P3–P4, which were appended with short sequence tails: C, TCGAG, AATTC, G, respectively. After gel-purification, the two PCR products were mixed together, denatured, and reannealed, resulting in 25% of the DNA fragments with Eco RI and XhoI overhangs. Concomitantly, the vector was double-digested with EcoRI and XhoI for 5–10 min at 37°C. After heat inactivation of the restriction enzyme at 95°C for 5 min, the vector was mixed with the reannealed JcDGAT2 containing EcoRI and XhoI overhangs, T4 DNA ligase and ATP were added and incubated at room temperature for 20 min, and finally transformed into E. coli . B, Restriction digestion (EcoRI and XhoI) of minipreps of pWXY1.0 (V) and pWXY1.0-JcDGAT2 (lane 1 to lane 10). The restriction pattern of pWXY1.0-JcDGAT2 generated by EcoRI and XhoI digestion was as predicted: 861 bp and 200 bp, respectively. M, DNA ruler DL2502 (Generay).

    Techniques Used: Clone Assay, Expressing, Plasmid Preparation, Amplification, Sequencing, Gel Purification, Polymerase Chain Reaction, Incubation, Transformation Assay, Generated

    Subcloning of one insert into a yeast expression vector pWXY1.0 by using IRDL cloning method. A, Schematic representation of one-step directional cloning of EGFP into a yeast expression vector pWXY1.0. B, Test of the IRDL cloning system. (1): Cloning of EGFP into pWXY1.0 by standard IRDL cloning step (the purified PCR products of GFP and yeast expression vector pWXY1.0 are mixed in a single tube together with FastDigest buffer, restriction enzymes XhoI and KpnI, ATP and T4 DNA ligase, and incubated at 37°C for 30 min), followed by transformation into E. coli Trans 1-T1 as described in materials and methods . (2): A control without T4 DNA ligase. (3): A control without restriction enzymes XhoI and KpnI. C, Colony PCR results from 48 recombinant colonies were run on 1% agarose gels. All of the colonies except the second clone tested contained the correct inserts. M, DNA ruler DL2501 from Generay. D, Plasmids DNA from 23 recombinant colonies and vector pWXY1.0 were digested with XhoI and KpnI and run on 1% agarose gels. DNA from all 23 recombinant colonies displayed the expected restriction pattern of pWXY1.0-EGFP. M, DNA ruler DL2502 (Generay). V, pWXY1.0.
    Figure Legend Snippet: Subcloning of one insert into a yeast expression vector pWXY1.0 by using IRDL cloning method. A, Schematic representation of one-step directional cloning of EGFP into a yeast expression vector pWXY1.0. B, Test of the IRDL cloning system. (1): Cloning of EGFP into pWXY1.0 by standard IRDL cloning step (the purified PCR products of GFP and yeast expression vector pWXY1.0 are mixed in a single tube together with FastDigest buffer, restriction enzymes XhoI and KpnI, ATP and T4 DNA ligase, and incubated at 37°C for 30 min), followed by transformation into E. coli Trans 1-T1 as described in materials and methods . (2): A control without T4 DNA ligase. (3): A control without restriction enzymes XhoI and KpnI. C, Colony PCR results from 48 recombinant colonies were run on 1% agarose gels. All of the colonies except the second clone tested contained the correct inserts. M, DNA ruler DL2501 from Generay. D, Plasmids DNA from 23 recombinant colonies and vector pWXY1.0 were digested with XhoI and KpnI and run on 1% agarose gels. DNA from all 23 recombinant colonies displayed the expected restriction pattern of pWXY1.0-EGFP. M, DNA ruler DL2502 (Generay). V, pWXY1.0.

    Techniques Used: Subcloning, Expressing, Plasmid Preparation, Clone Assay, Purification, Polymerase Chain Reaction, Incubation, Transformation Assay, Recombinant

    18) Product Images from "IRDL Cloning: A One-Tube, Zero-Background, Easy-to-Use, Directional Cloning Method Improves Throughput in Recombinant DNA Preparation"

    Article Title: IRDL Cloning: A One-Tube, Zero-Background, Easy-to-Use, Directional Cloning Method Improves Throughput in Recombinant DNA Preparation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0107907

    Schematic diagram of cloning the gene of interest containing internal restriction sites into expression vector. A, generation of sticky-end fragments and cloning into pWXY1.0 by IRDL cloning. The JcDGAT2 was amplified by two pairs of primers, P1–P2, and P3–P4, which were appended with short sequence tails: C, TCGAG, AATTC, G, respectively. After gel-purification, the two PCR products were mixed together, denatured, and reannealed, resulting in 25% of the DNA fragments with Eco RI and XhoI overhangs. Concomitantly, the vector was double-digested with EcoRI and XhoI for 5–10 min at 37°C. After heat inactivation of the restriction enzyme at 95°C for 5 min, the vector was mixed with the reannealed JcDGAT2 containing EcoRI and XhoI overhangs, T4 DNA ligase and ATP were added and incubated at room temperature for 20 min, and finally transformed into E. coli . B, Restriction digestion (EcoRI and XhoI) of minipreps of pWXY1.0 (V) and pWXY1.0-JcDGAT2 (lane 1 to lane 10). The restriction pattern of pWXY1.0-JcDGAT2 generated by EcoRI and XhoI digestion was as predicted: 861 bp and 200 bp, respectively. M, DNA ruler DL2502 (Generay).
    Figure Legend Snippet: Schematic diagram of cloning the gene of interest containing internal restriction sites into expression vector. A, generation of sticky-end fragments and cloning into pWXY1.0 by IRDL cloning. The JcDGAT2 was amplified by two pairs of primers, P1–P2, and P3–P4, which were appended with short sequence tails: C, TCGAG, AATTC, G, respectively. After gel-purification, the two PCR products were mixed together, denatured, and reannealed, resulting in 25% of the DNA fragments with Eco RI and XhoI overhangs. Concomitantly, the vector was double-digested with EcoRI and XhoI for 5–10 min at 37°C. After heat inactivation of the restriction enzyme at 95°C for 5 min, the vector was mixed with the reannealed JcDGAT2 containing EcoRI and XhoI overhangs, T4 DNA ligase and ATP were added and incubated at room temperature for 20 min, and finally transformed into E. coli . B, Restriction digestion (EcoRI and XhoI) of minipreps of pWXY1.0 (V) and pWXY1.0-JcDGAT2 (lane 1 to lane 10). The restriction pattern of pWXY1.0-JcDGAT2 generated by EcoRI and XhoI digestion was as predicted: 861 bp and 200 bp, respectively. M, DNA ruler DL2502 (Generay).

    Techniques Used: Clone Assay, Expressing, Plasmid Preparation, Amplification, Sequencing, Gel Purification, Polymerase Chain Reaction, Incubation, Transformation Assay, Generated

    Subcloning of one insert into a yeast expression vector pWXY1.0 by using IRDL cloning method. A, Schematic representation of one-step directional cloning of EGFP into a yeast expression vector pWXY1.0. B, Test of the IRDL cloning system. (1): Cloning of EGFP into pWXY1.0 by standard IRDL cloning step (the purified PCR products of GFP and yeast expression vector pWXY1.0 are mixed in a single tube together with FastDigest buffer, restriction enzymes XhoI and KpnI, ATP and T4 DNA ligase, and incubated at 37°C for 30 min), followed by transformation into E. coli Trans 1-T1 as described in materials and methods . (2): A control without T4 DNA ligase. (3): A control without restriction enzymes XhoI and KpnI. C, Colony PCR results from 48 recombinant colonies were run on 1% agarose gels. All of the colonies except the second clone tested contained the correct inserts. M, DNA ruler DL2501 from Generay. D, Plasmids DNA from 23 recombinant colonies and vector pWXY1.0 were digested with XhoI and KpnI and run on 1% agarose gels. DNA from all 23 recombinant colonies displayed the expected restriction pattern of pWXY1.0-EGFP. M, DNA ruler DL2502 (Generay). V, pWXY1.0.
    Figure Legend Snippet: Subcloning of one insert into a yeast expression vector pWXY1.0 by using IRDL cloning method. A, Schematic representation of one-step directional cloning of EGFP into a yeast expression vector pWXY1.0. B, Test of the IRDL cloning system. (1): Cloning of EGFP into pWXY1.0 by standard IRDL cloning step (the purified PCR products of GFP and yeast expression vector pWXY1.0 are mixed in a single tube together with FastDigest buffer, restriction enzymes XhoI and KpnI, ATP and T4 DNA ligase, and incubated at 37°C for 30 min), followed by transformation into E. coli Trans 1-T1 as described in materials and methods . (2): A control without T4 DNA ligase. (3): A control without restriction enzymes XhoI and KpnI. C, Colony PCR results from 48 recombinant colonies were run on 1% agarose gels. All of the colonies except the second clone tested contained the correct inserts. M, DNA ruler DL2501 from Generay. D, Plasmids DNA from 23 recombinant colonies and vector pWXY1.0 were digested with XhoI and KpnI and run on 1% agarose gels. DNA from all 23 recombinant colonies displayed the expected restriction pattern of pWXY1.0-EGFP. M, DNA ruler DL2502 (Generay). V, pWXY1.0.

    Techniques Used: Subcloning, Expressing, Plasmid Preparation, Clone Assay, Purification, Polymerase Chain Reaction, Incubation, Transformation Assay, Recombinant

    19) Product Images from "A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage"

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq756

    Principle, reaction efficiencies and NMR evidence for isotope labeling of each stem-loop of the RsmZ RNA separately. ( a ) Sequence-specific RNase H cleavages to obtain all four isotopically labeled stem-loop fragments. The yields of the cleavage reactions before HPLC purification are indicated, the values in brackets are expressing the yield after purification. The different stem-loops are colored (SL1: magenta, SL2: green, SL3: orange, SL4: cyan). ( b ) Splinted T4 DNA ligase mediated ligations of isotope labeled (in color) and unlabeled (in black) fragments. The unlabeled fragments were obtained in a similar way as the labeled fragments. ( c ) NMR evidence for the successful segmental isotope labeling of each stem-loop separately. 1 H- 15 N-HSQC NMR spectrum of the uniformly 15 N-labeled RsmZ RNA (left) and overlay of the 1 H- 15 N-HSQC NMR spectra of the four segmentally labeled RsmZ RNAs with each stem-loop labeled separately (right). The spectra were recorded on a Bruker 600 MHz spectrometer at 10°C.
    Figure Legend Snippet: Principle, reaction efficiencies and NMR evidence for isotope labeling of each stem-loop of the RsmZ RNA separately. ( a ) Sequence-specific RNase H cleavages to obtain all four isotopically labeled stem-loop fragments. The yields of the cleavage reactions before HPLC purification are indicated, the values in brackets are expressing the yield after purification. The different stem-loops are colored (SL1: magenta, SL2: green, SL3: orange, SL4: cyan). ( b ) Splinted T4 DNA ligase mediated ligations of isotope labeled (in color) and unlabeled (in black) fragments. The unlabeled fragments were obtained in a similar way as the labeled fragments. ( c ) NMR evidence for the successful segmental isotope labeling of each stem-loop separately. 1 H- 15 N-HSQC NMR spectrum of the uniformly 15 N-labeled RsmZ RNA (left) and overlay of the 1 H- 15 N-HSQC NMR spectra of the four segmentally labeled RsmZ RNAs with each stem-loop labeled separately (right). The spectra were recorded on a Bruker 600 MHz spectrometer at 10°C.

    Techniques Used: Nuclear Magnetic Resonance, Labeling, Sequencing, High Performance Liquid Chromatography, Purification, Expressing

    20) Product Images from "Establishing broad generality of DNA catalysts for site-specific hydrolysis of single-stranded DNA"

    Article Title: Establishing broad generality of DNA catalysts for site-specific hydrolysis of single-stranded DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr860

    Selection process that includes splint ligation pressure for DNA-catalyzed DNA hydrolysis at a particular predetermined site (X^G, where four different selections were performed for X = one of C, T, A, or G). The hydrolysis products are 3′-hydroxyl + 5′-phosphate products, as required by T4 DNA ligase. Note that the acceptor oligonucleotide, which has a 3′-hydroxyl for joining to the 5′-phosphate of the G nucleotide, has a long 5′-extension that leads to a large upward PAGE shift upon splint ligation. For nucleotide details, see the Experimental Section.
    Figure Legend Snippet: Selection process that includes splint ligation pressure for DNA-catalyzed DNA hydrolysis at a particular predetermined site (X^G, where four different selections were performed for X = one of C, T, A, or G). The hydrolysis products are 3′-hydroxyl + 5′-phosphate products, as required by T4 DNA ligase. Note that the acceptor oligonucleotide, which has a 3′-hydroxyl for joining to the 5′-phosphate of the G nucleotide, has a long 5′-extension that leads to a large upward PAGE shift upon splint ligation. For nucleotide details, see the Experimental Section.

    Techniques Used: Selection, Ligation, Polyacrylamide Gel Electrophoresis

    21) Product Images from "SPlinted Ligation Adapter Tagging (SPLAT), a novel library preparation method for whole genome bisulphite sequencing"

    Article Title: SPlinted Ligation Adapter Tagging (SPLAT), a novel library preparation method for whole genome bisulphite sequencing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1110

    Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard T4 DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.
    Figure Legend Snippet: Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard T4 DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.

    Techniques Used: Bisulfite Sequencing, Methylation, Construct, Amplification, Polymerase Chain Reaction, Next-Generation Sequencing, Sequencing, DNA Methylation Assay, Random Hexamer Labeling, DNA Ligation, Modification, Ligation

    22) Product Images from "An intact ribose moiety at A2602 of 23S rRNA is key to trigger peptidyl-tRNA hydrolysis during translation termination"

    Article Title: An intact ribose moiety at A2602 of 23S rRNA is key to trigger peptidyl-tRNA hydrolysis during translation termination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm539

    RF1-mediated peptide release activities of gapped-cp-reconstituted 50S subunits. (A) To measure peptidyl-tRNA hydrolysis activity, gapped-cp-reconstituted 50S (rec. 50S) were reassociated with native 30S subunits and programmed with an mRNA analog placing the stop codon UAA into the A-site. The peptidyl-tRNA analog f-Met-tRNA was bound to the P-site and the reaction initiated by the addition of RF1 from T. thermophilus . (B) The amount of hydrolyzed f-[ 3 H]Met-tRNA in the absence of ribosomal particles (reaction buffer), in the presence of the small ribosomal subunit (30S) or in the presence of 70S ribosomes containing native 30S and gapped-cp-reconstituted 50S after 30 min of incubation is shown. Gapped-cp-reconstituted 50S particles assembled from cp2623-2576 were tested in the absence and presence of the ligated 45-mer RNA fragment containing the wild-type sequence (+ wt oligo). 50S assembled form cp2623-2622 were assayed in the context of the wild-type sequence (cp2623-2622_wt) and in the context of the A2602 deletion mutant (cp2623-2622_Δ2602). RF1 was absent (−) or present (+) at a concentration of 0.8 µM. The mean and SD of released f-[ 3 H]Met from two representative experiments are shown (cpm) as well as the fraction of the 0.3 pmol input peptidyl-tRNA that was hydrolyzed are indicated. (C) Peptidyl-tRNA hydrolysis product yields of reconstituted 50S subunits that have been assembled from cp2623-2576 to which the compensating synthetic wild-type 45-mer RNA (wt-oligo) or the 2602C or 2602Δ variants have been ligated. The yield of f-[ 3 H]Met released by particles carrying the wt-oligo at the end of the reaction curve after 30 min of incubation has been taken as 1.00. The relative activities of gapped-cp-reconstituted 50S subunits carrying the ligated wt-oligo in the presence of antibiotics (sparsomycin: Spa; hygromycin A: HygA), or in the absence of an A-site-bound stop codon (no stop), as well as the activities of the two 2602 mutants are shown above the respective bars. The RF1-mediated hydrolysis strictly depends on the covalent connection of the synthetic RNA fragment to the cp-23S rRNA, since no product formation was observed in control experiments where the T4-DNA ligase has been omitted (no ligase). Values shown represent the mean and the SD of at least two independent experiments.
    Figure Legend Snippet: RF1-mediated peptide release activities of gapped-cp-reconstituted 50S subunits. (A) To measure peptidyl-tRNA hydrolysis activity, gapped-cp-reconstituted 50S (rec. 50S) were reassociated with native 30S subunits and programmed with an mRNA analog placing the stop codon UAA into the A-site. The peptidyl-tRNA analog f-Met-tRNA was bound to the P-site and the reaction initiated by the addition of RF1 from T. thermophilus . (B) The amount of hydrolyzed f-[ 3 H]Met-tRNA in the absence of ribosomal particles (reaction buffer), in the presence of the small ribosomal subunit (30S) or in the presence of 70S ribosomes containing native 30S and gapped-cp-reconstituted 50S after 30 min of incubation is shown. Gapped-cp-reconstituted 50S particles assembled from cp2623-2576 were tested in the absence and presence of the ligated 45-mer RNA fragment containing the wild-type sequence (+ wt oligo). 50S assembled form cp2623-2622 were assayed in the context of the wild-type sequence (cp2623-2622_wt) and in the context of the A2602 deletion mutant (cp2623-2622_Δ2602). RF1 was absent (−) or present (+) at a concentration of 0.8 µM. The mean and SD of released f-[ 3 H]Met from two representative experiments are shown (cpm) as well as the fraction of the 0.3 pmol input peptidyl-tRNA that was hydrolyzed are indicated. (C) Peptidyl-tRNA hydrolysis product yields of reconstituted 50S subunits that have been assembled from cp2623-2576 to which the compensating synthetic wild-type 45-mer RNA (wt-oligo) or the 2602C or 2602Δ variants have been ligated. The yield of f-[ 3 H]Met released by particles carrying the wt-oligo at the end of the reaction curve after 30 min of incubation has been taken as 1.00. The relative activities of gapped-cp-reconstituted 50S subunits carrying the ligated wt-oligo in the presence of antibiotics (sparsomycin: Spa; hygromycin A: HygA), or in the absence of an A-site-bound stop codon (no stop), as well as the activities of the two 2602 mutants are shown above the respective bars. The RF1-mediated hydrolysis strictly depends on the covalent connection of the synthetic RNA fragment to the cp-23S rRNA, since no product formation was observed in control experiments where the T4-DNA ligase has been omitted (no ligase). Values shown represent the mean and the SD of at least two independent experiments.

    Techniques Used: Activity Assay, Incubation, Sequencing, Mutagenesis, Concentration Assay

    23) Product Images from "Chromatin loop organization of the junb locus in mouse dendritic cells"

    Article Title: Chromatin loop organization of the junb locus in mouse dendritic cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt669

    Stronger transcriptional activity in response to LPS stimulation upon forced proximity of junb promoter and enhancer regions. ( A ) Transfection of mP-luc-E, E-mP-luc and Emut-mP-luc plasmids in DC2.4 cells. junb minimal promoter (mP), wild-type E domain and E-domain mutated on the NF-κB-responsive sites were cloned upstream or downstream of the luciferase gene ( luc ) of the pGL3 reporter plasmid as indicated in Aa. mP corresponds to positions −206/+31 in mouse junb and E to positions +2022/+2237. DC2.4 was transfected, stimulated and processed for luciferase assay as in Figure 2 G. Plasmids were cleaved with the Ase I restriction enzyme that cuts on both sides of the mP-luc-E, E-mP-luc and Emut-mP-luc fragments to avoid bias linked to the circular nature of plasmids. The presented data are the results of three independent experiments (Ab). ( B ) Transfection of linear and circular fragments bearing chimeric luc/junb genes . DNA fragments spanning the minimal junb promoter (starting at position −206) to the end of the E domain (position 2237) were purified from the p-junb-Luc- κ B- and the p-junb-Luc- κ Bmut reporter plasmids used in Figure 2 G. They were then circularized using the T4 DNA ligase as described in ‘Materials and Methods’ section. DC2.4 cells were then parallely transfected with the linear and circular isoforms of these fragments and LPS-stimulated as described in Ba before assays of both luciferase activity and luciferase DNA in cell lysates. The latter DNA assays showed comparable amounts of the DNA isoforms at the end of the experiments. The results of luciferase assay after normalization of data are presented in Bb. They correspond to four independent experiments. Details of experimental procedures are given in ‘Materials and Methods’ section.
    Figure Legend Snippet: Stronger transcriptional activity in response to LPS stimulation upon forced proximity of junb promoter and enhancer regions. ( A ) Transfection of mP-luc-E, E-mP-luc and Emut-mP-luc plasmids in DC2.4 cells. junb minimal promoter (mP), wild-type E domain and E-domain mutated on the NF-κB-responsive sites were cloned upstream or downstream of the luciferase gene ( luc ) of the pGL3 reporter plasmid as indicated in Aa. mP corresponds to positions −206/+31 in mouse junb and E to positions +2022/+2237. DC2.4 was transfected, stimulated and processed for luciferase assay as in Figure 2 G. Plasmids were cleaved with the Ase I restriction enzyme that cuts on both sides of the mP-luc-E, E-mP-luc and Emut-mP-luc fragments to avoid bias linked to the circular nature of plasmids. The presented data are the results of three independent experiments (Ab). ( B ) Transfection of linear and circular fragments bearing chimeric luc/junb genes . DNA fragments spanning the minimal junb promoter (starting at position −206) to the end of the E domain (position 2237) were purified from the p-junb-Luc- κ B- and the p-junb-Luc- κ Bmut reporter plasmids used in Figure 2 G. They were then circularized using the T4 DNA ligase as described in ‘Materials and Methods’ section. DC2.4 cells were then parallely transfected with the linear and circular isoforms of these fragments and LPS-stimulated as described in Ba before assays of both luciferase activity and luciferase DNA in cell lysates. The latter DNA assays showed comparable amounts of the DNA isoforms at the end of the experiments. The results of luciferase assay after normalization of data are presented in Bb. They correspond to four independent experiments. Details of experimental procedures are given in ‘Materials and Methods’ section.

    Techniques Used: Activity Assay, Transfection, Clone Assay, Luciferase, Plasmid Preparation, Purification

    24) Product Images from "Genomic Analysis of Pseudomonas putida Phage tf with Localized Single-Strand DNA Interruptions"

    Article Title: Genomic Analysis of Pseudomonas putida Phage tf with Localized Single-Strand DNA Interruptions

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0051163

    Primer extension analysis of the sci-14. The schematic of the experiment is shown at the top of the Figure: non-ligated (1) and ligated (2) DNA preparations were digested with HaeIII restriction endonuclease and primer extension reaction were performed using the same [ 32 P] labeled primer. The results of primer extension with Klenow fragment (PE:K) and Taq DNA polymerases (PE:T) are presented at the bottom part of the Figure. Bands corresponding to the stoppages at the sci-14 and Hae III sites are indicated by arrows. Sequencing ladders (A, G, C, T) were generated by Sanger sequencing of the T4 DNA ligase treated tf DNA employing the same [ 32 P] labeled primer used in primer extension.
    Figure Legend Snippet: Primer extension analysis of the sci-14. The schematic of the experiment is shown at the top of the Figure: non-ligated (1) and ligated (2) DNA preparations were digested with HaeIII restriction endonuclease and primer extension reaction were performed using the same [ 32 P] labeled primer. The results of primer extension with Klenow fragment (PE:K) and Taq DNA polymerases (PE:T) are presented at the bottom part of the Figure. Bands corresponding to the stoppages at the sci-14 and Hae III sites are indicated by arrows. Sequencing ladders (A, G, C, T) were generated by Sanger sequencing of the T4 DNA ligase treated tf DNA employing the same [ 32 P] labeled primer used in primer extension.

    Techniques Used: Labeling, Sequencing, Generated

    Identification and analysis of the localized nicks in tf DNA molecules. DNA preparation of bacteriophage tf was denatured in 0.1 M NaOH and subjected to electrophoresis in 0.9% agarose gel; in a control experiment a DNA from the same preparation was treated with T4 DNA ligase prior to denaturing in 0.1 M NaOH and electrophoresis.
    Figure Legend Snippet: Identification and analysis of the localized nicks in tf DNA molecules. DNA preparation of bacteriophage tf was denatured in 0.1 M NaOH and subjected to electrophoresis in 0.9% agarose gel; in a control experiment a DNA from the same preparation was treated with T4 DNA ligase prior to denaturing in 0.1 M NaOH and electrophoresis.

    Techniques Used: Electrophoresis, Agarose Gel Electrophoresis

    Analysis of the tf DNA ends. (A) Automated sequencing of the bacteriophage tf DNA at termini regions. Alternative end-nucleotides are indicated. (B) Results of the primer extension analysis of the 5′-ends in the tf DNA. Nucleotide positions corresponding to the stoppages at the phage DNA ends are indicated with arrows; black arrow for the position corresponding to the major band and gray arrows – position corresponding to minor bands at the left terminus only. (C) Analysis of the termini junction in the circularized tf DNA. Bacteriophage DNA was treated with T4 DNA polymerase and circularized using T4 DNA ligase. The junction region was analyzed by automated sequencing of the corresponding PCR product as described in the text. The junction identified is shown on the chromatogram by a vertical line and the schematic of the ligated genome ends is shown above the chromatogram. (D) Analysis of the 3′-ends in the tf DNA. Denatured tf DNA was treated with TdT enzyme in separate experiments in the presence of either dATP or dTTP and PCR products were obtained using resulting preparations and one phage specific and one poly-dT (poly-dA) specific primer. Chromatograms for all types of experiments are presented with the corresponding sequences shown at the bottom of the figure. Terminal sequences of the phage genome are shown by shading. (E) Structure of the tf DNA ends’ sequences.
    Figure Legend Snippet: Analysis of the tf DNA ends. (A) Automated sequencing of the bacteriophage tf DNA at termini regions. Alternative end-nucleotides are indicated. (B) Results of the primer extension analysis of the 5′-ends in the tf DNA. Nucleotide positions corresponding to the stoppages at the phage DNA ends are indicated with arrows; black arrow for the position corresponding to the major band and gray arrows – position corresponding to minor bands at the left terminus only. (C) Analysis of the termini junction in the circularized tf DNA. Bacteriophage DNA was treated with T4 DNA polymerase and circularized using T4 DNA ligase. The junction region was analyzed by automated sequencing of the corresponding PCR product as described in the text. The junction identified is shown on the chromatogram by a vertical line and the schematic of the ligated genome ends is shown above the chromatogram. (D) Analysis of the 3′-ends in the tf DNA. Denatured tf DNA was treated with TdT enzyme in separate experiments in the presence of either dATP or dTTP and PCR products were obtained using resulting preparations and one phage specific and one poly-dT (poly-dA) specific primer. Chromatograms for all types of experiments are presented with the corresponding sequences shown at the bottom of the figure. Terminal sequences of the phage genome are shown by shading. (E) Structure of the tf DNA ends’ sequences.

    Techniques Used: Sequencing, Polymerase Chain Reaction

    25) Product Images from "Highly specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification †Electronic supplementary information (ESI) available: Additional experimental materials, methods, DNA sequences and supplementary figures and tables. See DOI: 10.1039/c7sc00292kClick here for additional data file."

    Article Title: Highly specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification †Electronic supplementary information (ESI) available: Additional experimental materials, methods, DNA sequences and supplementary figures and tables. See DOI: 10.1039/c7sc00292kClick here for additional data file.

    Journal: Chemical Science

    doi: 10.1039/c7sc00292k

    Detecting target RNA in vitro using target RNA-initiated RCA with different ligases. (a) Fluorescence emission spectra for the target RNA-initiated RCA reaction carried out using different ligases (Splint R, T4 RNA ligase 2 and T4 DNA ligase) and padlock probes (Target, targeting mRNA ACTB; Random, random padlock probe). (b) The fluorescence intensity of the target RNA-initiated RCA reaction profiled in (a).
    Figure Legend Snippet: Detecting target RNA in vitro using target RNA-initiated RCA with different ligases. (a) Fluorescence emission spectra for the target RNA-initiated RCA reaction carried out using different ligases (Splint R, T4 RNA ligase 2 and T4 DNA ligase) and padlock probes (Target, targeting mRNA ACTB; Random, random padlock probe). (b) The fluorescence intensity of the target RNA-initiated RCA reaction profiled in (a).

    Techniques Used: In Vitro, Fluorescence

    Demonstration of the specificity for mRNA imaging in single cells. (a) The RCA reactions were carried using T4 DNA ligase, T4 RNA ligase 2 and Splint R. Four padlock probes were designed for target mRNA TK1: one perfectly matching with the target sequence of mRNA TK1 (Mis-0), two probes with one (Mis-1) or two (Mis-2) mismatching bases compared to target mRNA TK1 and one random probe (Ran). Inset: frequency histogram of RCA amplicons per cell detected. The right column is the average number of RCA amplicons detected in MCF-7 cells using the padlock probes Mis-0, Mis-1, Mis-2 and Ran; (b and c) detection of a single nucleotide difference in mRNA ACTB in human MCF-7 cells (b) and mouse 4T1 cells (c). Inset: the quantification of the average number of RCA amplicons detected (100 cells counted). The cell nuclei are shown in blue, the RCA amplicons appear as green or red spots, and the cell outlines are marked by a dotted line. Scale bars: 10 μm.
    Figure Legend Snippet: Demonstration of the specificity for mRNA imaging in single cells. (a) The RCA reactions were carried using T4 DNA ligase, T4 RNA ligase 2 and Splint R. Four padlock probes were designed for target mRNA TK1: one perfectly matching with the target sequence of mRNA TK1 (Mis-0), two probes with one (Mis-1) or two (Mis-2) mismatching bases compared to target mRNA TK1 and one random probe (Ran). Inset: frequency histogram of RCA amplicons per cell detected. The right column is the average number of RCA amplicons detected in MCF-7 cells using the padlock probes Mis-0, Mis-1, Mis-2 and Ran; (b and c) detection of a single nucleotide difference in mRNA ACTB in human MCF-7 cells (b) and mouse 4T1 cells (c). Inset: the quantification of the average number of RCA amplicons detected (100 cells counted). The cell nuclei are shown in blue, the RCA amplicons appear as green or red spots, and the cell outlines are marked by a dotted line. Scale bars: 10 μm.

    Techniques Used: Imaging, Sequencing

    Effect of different ligases on the efficiency of mRNA imaging in single cells. (a) Imaging of mRNA ACTB using target RNA-initiated RCA in the MCF-7 cells with different ligases: T4 DNA ligase, T4 RNA ligase 2 and Splint R. In situ RCA was conducted using the padlock probe targeting ACTB and a random padlock probe as control. The cell nuclei are shown in blue, the RCA amplicons appear as green spots, and the cell outlines are marked by a dotted line. Scale bars: 10 μm. Inset: frequency histogram of RCA amplicons per cell detected. (b) Quantification of the average number of RCA amplicons per cell detected in (a).
    Figure Legend Snippet: Effect of different ligases on the efficiency of mRNA imaging in single cells. (a) Imaging of mRNA ACTB using target RNA-initiated RCA in the MCF-7 cells with different ligases: T4 DNA ligase, T4 RNA ligase 2 and Splint R. In situ RCA was conducted using the padlock probe targeting ACTB and a random padlock probe as control. The cell nuclei are shown in blue, the RCA amplicons appear as green spots, and the cell outlines are marked by a dotted line. Scale bars: 10 μm. Inset: frequency histogram of RCA amplicons per cell detected. (b) Quantification of the average number of RCA amplicons per cell detected in (a).

    Techniques Used: Imaging, In Situ

    26) Product Images from "Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase"

    Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx033

    Single-stranded DNA ligation with T4 DNA ligase and CircLigase. A pool of 60 nt acceptor oligonucleotides (‘60N’) were ligated to 10 pmol of a 3΄ biotinylated donor oligonucleotide (CL78) using either T4 DNA ligase in the presence of a splinter oligonucleotide (TL38) or CircLigase. Ligation products were visualized on a 10% denaturing polyacrylamide gel stained with SybrGold. Band shifts from 60 nt to 80 nt indicate successful ligation. Schematic overviews of the reaction schemes are shown on top. The scheme developed by Kwok et al . ( 19 ) is shown for comparison. M: Single-stranded DNA size marker.
    Figure Legend Snippet: Single-stranded DNA ligation with T4 DNA ligase and CircLigase. A pool of 60 nt acceptor oligonucleotides (‘60N’) were ligated to 10 pmol of a 3΄ biotinylated donor oligonucleotide (CL78) using either T4 DNA ligase in the presence of a splinter oligonucleotide (TL38) or CircLigase. Ligation products were visualized on a 10% denaturing polyacrylamide gel stained with SybrGold. Band shifts from 60 nt to 80 nt indicate successful ligation. Schematic overviews of the reaction schemes are shown on top. The scheme developed by Kwok et al . ( 19 ) is shown for comparison. M: Single-stranded DNA size marker.

    Techniques Used: DNA Ligation, Ligation, Staining, Marker

    Library preparation methods for highly degraded DNA. ( A ) In the single-stranded library preparation method described here (ssDNA2.0), DNA fragments (black) are 5΄ and 3΄ dephosphorylated and separated into single strands by heat denaturation. 3΄ biotinylated adapter molecules (red) are attached to the 3΄ ends of the DNA fragments via hybridization to a stretch of six random nucleotides (marked as ‘N’) belonging to a splinter oligonucleotide complementary to the adapter and nick closure with T4 DNA ligase. Following the immobilization of the ligation products on streptavidin-coated beads, the splinter oligonucleotide is removed by bead wash at an elevated temperature. Synthesis of the second strand is carried out using the Klenow fragment of Escherichia coli DNA polymerase I and a primer with phosphorothioate backbone modifications (red stars) to prevent exonucleolytic degradation. Unincorporated primers are removed through a bead wash at an elevated temperature, preventing the formation of adapter dimers in the subsequent blunt-end ligation reaction, which is again catalyzed by T4 DNA ligase. Adapter self-ligation is prevented through a 3΄ dideoxy modification in the adapter. The final library strand is released from the beads by heat denaturation. ( B ) In the single-stranded library preparation method originally described in Gansauge and Meyer, ( 4 ), the first adapter was attached through true single-stranded DNA ligation using CircLigase. The large fragment of Bst DNA polymerase was used to copy the template strand, leaving overhanging 3΄ nucleotides, which had to be removed in a blunt-end repair reaction using T4 DNA polymerase. ( C ) The ‘454’ method of double-stranded library preparation in the implementation of Meyer and Kircher, ( 23 ), is based on non-directional blunt-end ligation of a mixture of two adapters to blunt-end repaired DNA fragments using T4 DNA ligase. To prevent adapter self-ligation, no phosphate groups are present at the 5΄ ends of the adapters, resulting in the ligation of the adapter strands only and necessitating subsequent nick fill-in with a strand-displacing polymerase. Intermittent DNA purification steps are required in-between enzymatic reactions. ( D ) The ‘Illumina’ method of double-stranded library preparation, shown here as implemented in New England Biolabs’ NEBNext Ultra II kit, requires the addition of A-overhangs (marked as ‘A’) to blunt-end repaired DNA fragments using a 3΄-5΄ exonuclease deletion mutant of the Klenow fragment of E. coli DNA polymerase I. Both adapter sequences are combined into one bell-shaped structure, which carries a 3΄ T overhang to allow sticky end ligation with T4 DNA ligase. Following ligation, adapter strands are separated by excision of uracil. Excess adapters and adapter dimers are removed through size-selective purification.
    Figure Legend Snippet: Library preparation methods for highly degraded DNA. ( A ) In the single-stranded library preparation method described here (ssDNA2.0), DNA fragments (black) are 5΄ and 3΄ dephosphorylated and separated into single strands by heat denaturation. 3΄ biotinylated adapter molecules (red) are attached to the 3΄ ends of the DNA fragments via hybridization to a stretch of six random nucleotides (marked as ‘N’) belonging to a splinter oligonucleotide complementary to the adapter and nick closure with T4 DNA ligase. Following the immobilization of the ligation products on streptavidin-coated beads, the splinter oligonucleotide is removed by bead wash at an elevated temperature. Synthesis of the second strand is carried out using the Klenow fragment of Escherichia coli DNA polymerase I and a primer with phosphorothioate backbone modifications (red stars) to prevent exonucleolytic degradation. Unincorporated primers are removed through a bead wash at an elevated temperature, preventing the formation of adapter dimers in the subsequent blunt-end ligation reaction, which is again catalyzed by T4 DNA ligase. Adapter self-ligation is prevented through a 3΄ dideoxy modification in the adapter. The final library strand is released from the beads by heat denaturation. ( B ) In the single-stranded library preparation method originally described in Gansauge and Meyer, ( 4 ), the first adapter was attached through true single-stranded DNA ligation using CircLigase. The large fragment of Bst DNA polymerase was used to copy the template strand, leaving overhanging 3΄ nucleotides, which had to be removed in a blunt-end repair reaction using T4 DNA polymerase. ( C ) The ‘454’ method of double-stranded library preparation in the implementation of Meyer and Kircher, ( 23 ), is based on non-directional blunt-end ligation of a mixture of two adapters to blunt-end repaired DNA fragments using T4 DNA ligase. To prevent adapter self-ligation, no phosphate groups are present at the 5΄ ends of the adapters, resulting in the ligation of the adapter strands only and necessitating subsequent nick fill-in with a strand-displacing polymerase. Intermittent DNA purification steps are required in-between enzymatic reactions. ( D ) The ‘Illumina’ method of double-stranded library preparation, shown here as implemented in New England Biolabs’ NEBNext Ultra II kit, requires the addition of A-overhangs (marked as ‘A’) to blunt-end repaired DNA fragments using a 3΄-5΄ exonuclease deletion mutant of the Klenow fragment of E. coli DNA polymerase I. Both adapter sequences are combined into one bell-shaped structure, which carries a 3΄ T overhang to allow sticky end ligation with T4 DNA ligase. Following ligation, adapter strands are separated by excision of uracil. Excess adapters and adapter dimers are removed through size-selective purification.

    Techniques Used: Hybridization, Ligation, Modification, DNA Ligation, DNA Purification, Mutagenesis, Purification

    Effects of single-stranded ligation schemes on library characteristics. ( A ) Informative sequence content of libraries prepared with CircLigase and T4 DNA ligase as a function of the input volume of ancient DNA extract used for library preparation. ( B ) Average GC content of the sequences obtained with the two ligation schemes. Note that the average GC content exceeds that of a typical mammalian genome because most sequences derive from microbial DNA, which is the dominant source of DNA in most ancient bones. ( C ) Fragment size distribution in the libraries as inferred from overlap-merged paired-end reads. Short artifacts in the library prepared from extremely little input DNA (corresponding to ∼1 mg bone) are mainly due to the incorporation of splinter fragments. ( D ) Frequencies of damage-induced C to T substitutions near the 5΄ and 3΄ ends of sequences.
    Figure Legend Snippet: Effects of single-stranded ligation schemes on library characteristics. ( A ) Informative sequence content of libraries prepared with CircLigase and T4 DNA ligase as a function of the input volume of ancient DNA extract used for library preparation. ( B ) Average GC content of the sequences obtained with the two ligation schemes. Note that the average GC content exceeds that of a typical mammalian genome because most sequences derive from microbial DNA, which is the dominant source of DNA in most ancient bones. ( C ) Fragment size distribution in the libraries as inferred from overlap-merged paired-end reads. Short artifacts in the library prepared from extremely little input DNA (corresponding to ∼1 mg bone) are mainly due to the incorporation of splinter fragments. ( D ) Frequencies of damage-induced C to T substitutions near the 5΄ and 3΄ ends of sequences.

    Techniques Used: Ligation, Sequencing, Ancient DNA Assay

    27) Product Images from "Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase"

    Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx033

    Single-stranded DNA ligation with T4 DNA ligase and CircLigase. A pool of 60 nt acceptor oligonucleotides (‘60N’) were ligated to 10 pmol of a 3΄ biotinylated donor oligonucleotide (CL78) using either T4 DNA ligase in the presence of a splinter oligonucleotide (TL38) or CircLigase. Ligation products were visualized on a 10% denaturing polyacrylamide gel stained with SybrGold. Band shifts from 60 nt to 80 nt indicate successful ligation. Schematic overviews of the reaction schemes are shown on top. The scheme developed by Kwok et al . ( 19 ) is shown for comparison. M: Single-stranded DNA size marker.
    Figure Legend Snippet: Single-stranded DNA ligation with T4 DNA ligase and CircLigase. A pool of 60 nt acceptor oligonucleotides (‘60N’) were ligated to 10 pmol of a 3΄ biotinylated donor oligonucleotide (CL78) using either T4 DNA ligase in the presence of a splinter oligonucleotide (TL38) or CircLigase. Ligation products were visualized on a 10% denaturing polyacrylamide gel stained with SybrGold. Band shifts from 60 nt to 80 nt indicate successful ligation. Schematic overviews of the reaction schemes are shown on top. The scheme developed by Kwok et al . ( 19 ) is shown for comparison. M: Single-stranded DNA size marker.

    Techniques Used: DNA Ligation, Ligation, Staining, Marker

    Library preparation methods for highly degraded DNA. ( A ) In the single-stranded library preparation method described here (ssDNA2.0), DNA fragments (black) are 5΄ and 3΄ dephosphorylated and separated into single strands by heat denaturation. 3΄ biotinylated adapter molecules (red) are attached to the 3΄ ends of the DNA fragments via hybridization to a stretch of six random nucleotides (marked as ‘N’) belonging to a splinter oligonucleotide complementary to the adapter and nick closure with T4 DNA ligase. Following the immobilization of the ligation products on streptavidin-coated beads, the splinter oligonucleotide is removed by bead wash at an elevated temperature. Synthesis of the second strand is carried out using the Klenow fragment of Escherichia coli DNA polymerase I and a primer with phosphorothioate backbone modifications (red stars) to prevent exonucleolytic degradation. Unincorporated primers are removed through a bead wash at an elevated temperature, preventing the formation of adapter dimers in the subsequent blunt-end ligation reaction, which is again catalyzed by T4 DNA ligase. Adapter self-ligation is prevented through a 3΄ dideoxy modification in the adapter. The final library strand is released from the beads by heat denaturation. ( B ) In the single-stranded library preparation method originally described in Gansauge and Meyer, ( 4 ), the first adapter was attached through true single-stranded DNA ligation using CircLigase. The large fragment of Bst DNA polymerase was used to copy the template strand, leaving overhanging 3΄ nucleotides, which had to be removed in a blunt-end repair reaction using T4 DNA polymerase. ( C ) The ‘454’ method of double-stranded library preparation in the implementation of Meyer and Kircher, ( 23 ), is based on non-directional blunt-end ligation of a mixture of two adapters to blunt-end repaired DNA fragments using T4 DNA ligase. To prevent adapter self-ligation, no phosphate groups are present at the 5΄ ends of the adapters, resulting in the ligation of the adapter strands only and necessitating subsequent nick fill-in with a strand-displacing polymerase. Intermittent DNA purification steps are required in-between enzymatic reactions. ( D ) The ‘Illumina’ method of double-stranded library preparation, shown here as implemented in New England Biolabs’ NEBNext Ultra II kit, requires the addition of A-overhangs (marked as ‘A’) to blunt-end repaired DNA fragments using a 3΄-5΄ exonuclease deletion mutant of the Klenow fragment of E. coli DNA polymerase I. Both adapter sequences are combined into one bell-shaped structure, which carries a 3΄ T overhang to allow sticky end ligation with T4 DNA ligase. Following ligation, adapter strands are separated by excision of uracil. Excess adapters and adapter dimers are removed through size-selective purification.
    Figure Legend Snippet: Library preparation methods for highly degraded DNA. ( A ) In the single-stranded library preparation method described here (ssDNA2.0), DNA fragments (black) are 5΄ and 3΄ dephosphorylated and separated into single strands by heat denaturation. 3΄ biotinylated adapter molecules (red) are attached to the 3΄ ends of the DNA fragments via hybridization to a stretch of six random nucleotides (marked as ‘N’) belonging to a splinter oligonucleotide complementary to the adapter and nick closure with T4 DNA ligase. Following the immobilization of the ligation products on streptavidin-coated beads, the splinter oligonucleotide is removed by bead wash at an elevated temperature. Synthesis of the second strand is carried out using the Klenow fragment of Escherichia coli DNA polymerase I and a primer with phosphorothioate backbone modifications (red stars) to prevent exonucleolytic degradation. Unincorporated primers are removed through a bead wash at an elevated temperature, preventing the formation of adapter dimers in the subsequent blunt-end ligation reaction, which is again catalyzed by T4 DNA ligase. Adapter self-ligation is prevented through a 3΄ dideoxy modification in the adapter. The final library strand is released from the beads by heat denaturation. ( B ) In the single-stranded library preparation method originally described in Gansauge and Meyer, ( 4 ), the first adapter was attached through true single-stranded DNA ligation using CircLigase. The large fragment of Bst DNA polymerase was used to copy the template strand, leaving overhanging 3΄ nucleotides, which had to be removed in a blunt-end repair reaction using T4 DNA polymerase. ( C ) The ‘454’ method of double-stranded library preparation in the implementation of Meyer and Kircher, ( 23 ), is based on non-directional blunt-end ligation of a mixture of two adapters to blunt-end repaired DNA fragments using T4 DNA ligase. To prevent adapter self-ligation, no phosphate groups are present at the 5΄ ends of the adapters, resulting in the ligation of the adapter strands only and necessitating subsequent nick fill-in with a strand-displacing polymerase. Intermittent DNA purification steps are required in-between enzymatic reactions. ( D ) The ‘Illumina’ method of double-stranded library preparation, shown here as implemented in New England Biolabs’ NEBNext Ultra II kit, requires the addition of A-overhangs (marked as ‘A’) to blunt-end repaired DNA fragments using a 3΄-5΄ exonuclease deletion mutant of the Klenow fragment of E. coli DNA polymerase I. Both adapter sequences are combined into one bell-shaped structure, which carries a 3΄ T overhang to allow sticky end ligation with T4 DNA ligase. Following ligation, adapter strands are separated by excision of uracil. Excess adapters and adapter dimers are removed through size-selective purification.

    Techniques Used: Hybridization, Ligation, Modification, DNA Ligation, DNA Purification, Mutagenesis, Purification

    Effects of single-stranded ligation schemes on library characteristics. ( A ) Informative sequence content of libraries prepared with CircLigase and T4 DNA ligase as a function of the input volume of ancient DNA extract used for library preparation. ( B ) Average GC content of the sequences obtained with the two ligation schemes. Note that the average GC content exceeds that of a typical mammalian genome because most sequences derive from microbial DNA, which is the dominant source of DNA in most ancient bones. ( C ) Fragment size distribution in the libraries as inferred from overlap-merged paired-end reads. Short artifacts in the library prepared from extremely little input DNA (corresponding to ∼1 mg bone) are mainly due to the incorporation of splinter fragments. ( D ) Frequencies of damage-induced C to T substitutions near the 5΄ and 3΄ ends of sequences.
    Figure Legend Snippet: Effects of single-stranded ligation schemes on library characteristics. ( A ) Informative sequence content of libraries prepared with CircLigase and T4 DNA ligase as a function of the input volume of ancient DNA extract used for library preparation. ( B ) Average GC content of the sequences obtained with the two ligation schemes. Note that the average GC content exceeds that of a typical mammalian genome because most sequences derive from microbial DNA, which is the dominant source of DNA in most ancient bones. ( C ) Fragment size distribution in the libraries as inferred from overlap-merged paired-end reads. Short artifacts in the library prepared from extremely little input DNA (corresponding to ∼1 mg bone) are mainly due to the incorporation of splinter fragments. ( D ) Frequencies of damage-induced C to T substitutions near the 5΄ and 3΄ ends of sequences.

    Techniques Used: Ligation, Sequencing, Ancient DNA Assay

    28) Product Images from "A new enzymatic route for production of long 5'-phosphorylated oligonucleotides using suicide cassettes and rolling circle DNA synthesis"

    Article Title: A new enzymatic route for production of long 5'-phosphorylated oligonucleotides using suicide cassettes and rolling circle DNA synthesis

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-7-49

    Solid support rolling circle DNA synthesis from amplified SF-WT90 oligonucleotide cleaved with Mly I . First row : The chemically synthesized padlock probe, WT90-66b, was incubated with a covalently coupled primer (Amin-L16-Mly I (+) primer or Amin-L16-Mly I (-)) in the presence of T4 DNA ligase. Only correctly hybridized padlock probes of the right sequence can be circularized. Ligated probes were subsequently amplified by rolling circle DNA synthesis and detected by hybridization to the rolling circle products of either the ID 16 or the anti ID 16 detection oligonucleotide. Second row : The SF-WT90 oligonucleotide, was amplified one round by the method presented in figure 1 (from (+)-strand to (-)-strand) and the suicide cassette was removed by cleavage with Mly I. Subsequently, the padlock probe generated was incubated with a covalently coupled primer (Amin-L16-Mly I (+) or Amin-L16- Mly I (-)) in the presence of T4 DNA ligase. Only correctly hybridized padlock probes of the right sequence can be circularized. Ligated probes were subsequently amplified by rolling circle DNA synthesis and detected by hybridization to the rolling circle products of either the ID 16 or the anti ID 16 detection oligonucleotide. Third row : The SF-WT90oligonucleotide was amplified two rounds by the method presented in figure 1 (from (+)-strand to (-)-strand and back to (+)-strand) and the suicide cassette was removed by cleavage with Mly I. Subsequently, the padlock probe generated was incubated with a covalently coupled primer (Amin-L16-Mly I (+) primer or Amin-L16- Mly I (-)) in the presence of T4 DNA ligase. Only correctly hybridized padlock probes of the right sequence can be circularized. Ligated probes were subsequently amplified by rolling circle DNA synthesis and detected by hybridization to the rolling circle products of either the ID 16 or the anti ID 16 detection oligonucleotide. Schematic representations indicate the expected outcome of the hybridization events. (+) and (-) indicate the polarity of the probes. The (+)-primer hybridizes to the (+)-probe and the (-)-primer hybridizes to the (-)-probe. Equimolar amounts of probe were applied in each reaction (0.1 μM). Scale bar, 100 μm. At the bottom of the figure is a schematic representation of the individual steps in the solid support assay.
    Figure Legend Snippet: Solid support rolling circle DNA synthesis from amplified SF-WT90 oligonucleotide cleaved with Mly I . First row : The chemically synthesized padlock probe, WT90-66b, was incubated with a covalently coupled primer (Amin-L16-Mly I (+) primer or Amin-L16-Mly I (-)) in the presence of T4 DNA ligase. Only correctly hybridized padlock probes of the right sequence can be circularized. Ligated probes were subsequently amplified by rolling circle DNA synthesis and detected by hybridization to the rolling circle products of either the ID 16 or the anti ID 16 detection oligonucleotide. Second row : The SF-WT90 oligonucleotide, was amplified one round by the method presented in figure 1 (from (+)-strand to (-)-strand) and the suicide cassette was removed by cleavage with Mly I. Subsequently, the padlock probe generated was incubated with a covalently coupled primer (Amin-L16-Mly I (+) or Amin-L16- Mly I (-)) in the presence of T4 DNA ligase. Only correctly hybridized padlock probes of the right sequence can be circularized. Ligated probes were subsequently amplified by rolling circle DNA synthesis and detected by hybridization to the rolling circle products of either the ID 16 or the anti ID 16 detection oligonucleotide. Third row : The SF-WT90oligonucleotide was amplified two rounds by the method presented in figure 1 (from (+)-strand to (-)-strand and back to (+)-strand) and the suicide cassette was removed by cleavage with Mly I. Subsequently, the padlock probe generated was incubated with a covalently coupled primer (Amin-L16-Mly I (+) primer or Amin-L16- Mly I (-)) in the presence of T4 DNA ligase. Only correctly hybridized padlock probes of the right sequence can be circularized. Ligated probes were subsequently amplified by rolling circle DNA synthesis and detected by hybridization to the rolling circle products of either the ID 16 or the anti ID 16 detection oligonucleotide. Schematic representations indicate the expected outcome of the hybridization events. (+) and (-) indicate the polarity of the probes. The (+)-primer hybridizes to the (+)-probe and the (-)-primer hybridizes to the (-)-probe. Equimolar amounts of probe were applied in each reaction (0.1 μM). Scale bar, 100 μm. At the bottom of the figure is a schematic representation of the individual steps in the solid support assay.

    Techniques Used: DNA Synthesis, Amplification, Synthesized, Incubation, Sequencing, Hybridization, Generated

    Comparison of the chemically synthesized oligonucleotide WT90-66b and the oligonucleotide contained within the suicide cassette in SF-WT90 following amplification in a solid support rolling circle DNA synthesis assay . The chemically synthesized padlock probe WT90-66b (left) and SF-WT90 (amplified two rounds (from (+)-strand to (-)-strand and back to (+)-strand) and cleaved with Mly I; the probe was purified by PAGE after each round) (right) were incubated with a covalently coupled primer (Amin-L16-Mly I (+)) in the presence of T4 DNA ligase. Only correctly hybridized padlock probes of the right sequence can be circularized. Ligated probes were subsequently amplified by rolling circle DNA synthesis and detected by hybridization to the rolling circle products of the ID 16 detection oligonucleotide. Equimolar amounts of probe were applied in each reaction (0.1 nM). Scale bar, 100 μm.
    Figure Legend Snippet: Comparison of the chemically synthesized oligonucleotide WT90-66b and the oligonucleotide contained within the suicide cassette in SF-WT90 following amplification in a solid support rolling circle DNA synthesis assay . The chemically synthesized padlock probe WT90-66b (left) and SF-WT90 (amplified two rounds (from (+)-strand to (-)-strand and back to (+)-strand) and cleaved with Mly I; the probe was purified by PAGE after each round) (right) were incubated with a covalently coupled primer (Amin-L16-Mly I (+)) in the presence of T4 DNA ligase. Only correctly hybridized padlock probes of the right sequence can be circularized. Ligated probes were subsequently amplified by rolling circle DNA synthesis and detected by hybridization to the rolling circle products of the ID 16 detection oligonucleotide. Equimolar amounts of probe were applied in each reaction (0.1 nM). Scale bar, 100 μm.

    Techniques Used: Synthesized, Amplification, DNA Synthesis, Purification, Polyacrylamide Gel Electrophoresis, Incubation, Sequencing, Hybridization

    29) Product Images from "Mutants of phage bIL67 RuvC with enhanced Holliday junction binding selectivity and resolution symmetry"

    Article Title: Mutants of phage bIL67 RuvC with enhanced Holliday junction binding selectivity and resolution symmetry

    Journal: Molecular Microbiology

    doi: 10.1111/mmi.12343

    Sequence specificity and symmetry of cleavage by 67RuvC R121A and R124A mutant proteins. A. Mapping of R121A, R124A and wt 67RuvC cleavage sites on junction (J11 and J12) and fork (F11 and F12) DNA substrates. Reactions contained 10 mM MgCl 2 , 0.6 nM 32 P-labelled DNA and protein at 10 nM. Lanes a, e, i and m served as no protein controls. Reactions were incubated for 15 min at 37°C before separation on 10% denaturing PAGE. B. Location of incisions on the 32 P-labelled strand of each substrate. The homologous core of junctions J11 and J12 are indicated in black flanked by heterologous sequences in grey; in the F11 and F12 fork substrates this core homology is also highlighted despite being fully annealed to its complement at one end. Cut sites were located by comparison with previous mapping data with wt 67RuvC (Curtis et al ., 2005 ) and are indicated by triangles, with the intensity of shading proportional to the amount of cleavage at a particular position. Asterisks indicate major asymmetrical incisions made by wt 67RuvC lying outside the region of homology in J11 and J12 (Curtis et al ., 2005 ). C. Products of cleavage by R121A, R124A and wt 67RuvC on junction (J11 and J12) and fork (F11 and F12) DNA substrates. Reactions were performed as in (A) with DNA separated on a 10% neutral gel to visualize the products of cleavage. D. Ligation of the products of R121A, R124A and wt 67RuvC Holliday junction resolution. Reactions contained 10 mM MgCl 2 , 0.6 nM 32 P-labelled J12 and 40 nM protein and were incubated at 37°C for 15 min. ATP (1 mM) was added and half of each reaction transferred to a fresh tube. T4 DNA ligase (2.5 units) was added to one half of each reaction and incubation at 37°C continued for a further 15 min. Samples were separated on a 10% denaturing polyacrylamide gel and analysed by phosphorimaging. ImageJ was used to quantify the amount of ligation at the four major sites cleaved by wt 67RuvC. Values are expressed as a percentage of the total radioactivity units detected in each lane in the presence (+) or absence (−) of DNA ligase.
    Figure Legend Snippet: Sequence specificity and symmetry of cleavage by 67RuvC R121A and R124A mutant proteins. A. Mapping of R121A, R124A and wt 67RuvC cleavage sites on junction (J11 and J12) and fork (F11 and F12) DNA substrates. Reactions contained 10 mM MgCl 2 , 0.6 nM 32 P-labelled DNA and protein at 10 nM. Lanes a, e, i and m served as no protein controls. Reactions were incubated for 15 min at 37°C before separation on 10% denaturing PAGE. B. Location of incisions on the 32 P-labelled strand of each substrate. The homologous core of junctions J11 and J12 are indicated in black flanked by heterologous sequences in grey; in the F11 and F12 fork substrates this core homology is also highlighted despite being fully annealed to its complement at one end. Cut sites were located by comparison with previous mapping data with wt 67RuvC (Curtis et al ., 2005 ) and are indicated by triangles, with the intensity of shading proportional to the amount of cleavage at a particular position. Asterisks indicate major asymmetrical incisions made by wt 67RuvC lying outside the region of homology in J11 and J12 (Curtis et al ., 2005 ). C. Products of cleavage by R121A, R124A and wt 67RuvC on junction (J11 and J12) and fork (F11 and F12) DNA substrates. Reactions were performed as in (A) with DNA separated on a 10% neutral gel to visualize the products of cleavage. D. Ligation of the products of R121A, R124A and wt 67RuvC Holliday junction resolution. Reactions contained 10 mM MgCl 2 , 0.6 nM 32 P-labelled J12 and 40 nM protein and were incubated at 37°C for 15 min. ATP (1 mM) was added and half of each reaction transferred to a fresh tube. T4 DNA ligase (2.5 units) was added to one half of each reaction and incubation at 37°C continued for a further 15 min. Samples were separated on a 10% denaturing polyacrylamide gel and analysed by phosphorimaging. ImageJ was used to quantify the amount of ligation at the four major sites cleaved by wt 67RuvC. Values are expressed as a percentage of the total radioactivity units detected in each lane in the presence (+) or absence (−) of DNA ligase.

    Techniques Used: Sequencing, Mutagenesis, Incubation, Polyacrylamide Gel Electrophoresis, Ligation, Radioactivity

    30) Product Images from "Regulation by interdomain communication of a headful packaging nuclease from bacteriophage T4"

    Article Title: Regulation by interdomain communication of a headful packaging nuclease from bacteriophage T4

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1191

    gp17 nuclease prefers long DNA substrates and cleaves at the ends of linear DNA. ( A ) Increasing concentrations of gp17 were incubated with 0.9 nM each of 29 kb pAd10 plasmid DNA or 2.6 kb pUC19 plasmid DNA. The undigested circular DNA was quantified and used to determine the percent of cleaved DNA at different gp17:DNA ratios. Values represent the average of duplicates from two independent experiments. ( B ) gp17 preference for longer DNA molecules was seen by incubating gp17 (3 µM, lanes 2–7) with a 2-log DNA ladder (400 ng, 0.1–10 kb, New England Biolabs) for 2–30 min. ( C ) Autoradiogram showing the cleavage of γ 32 P end-labeled λ-HindIII DNA fragments (0.5 pmol, 125–23 130 bp, Promega) by gp17 (1.2 µM) (lanes 2–6) or DNase I (0.0024 µM, 500-fold less than gp17) (lanes 7–11). Lane 1 has untreated DNA. ( D ) gp17 nuclease generates blunt ends. Circular pUC19 DNA (40 ng) was cleaved by gp17 (lanes 2–4) or BamH1 (lanes 5–7). The cleaved DNA was then treated with E. coli DNA ligase (lanes 3 and 6) or T4 DNA ligase (lanes 4 and 7). Lanes labeled as ‘C’ are control untreated lanes. See ‘Materials and Methods’ section for additional details.
    Figure Legend Snippet: gp17 nuclease prefers long DNA substrates and cleaves at the ends of linear DNA. ( A ) Increasing concentrations of gp17 were incubated with 0.9 nM each of 29 kb pAd10 plasmid DNA or 2.6 kb pUC19 plasmid DNA. The undigested circular DNA was quantified and used to determine the percent of cleaved DNA at different gp17:DNA ratios. Values represent the average of duplicates from two independent experiments. ( B ) gp17 preference for longer DNA molecules was seen by incubating gp17 (3 µM, lanes 2–7) with a 2-log DNA ladder (400 ng, 0.1–10 kb, New England Biolabs) for 2–30 min. ( C ) Autoradiogram showing the cleavage of γ 32 P end-labeled λ-HindIII DNA fragments (0.5 pmol, 125–23 130 bp, Promega) by gp17 (1.2 µM) (lanes 2–6) or DNase I (0.0024 µM, 500-fold less than gp17) (lanes 7–11). Lane 1 has untreated DNA. ( D ) gp17 nuclease generates blunt ends. Circular pUC19 DNA (40 ng) was cleaved by gp17 (lanes 2–4) or BamH1 (lanes 5–7). The cleaved DNA was then treated with E. coli DNA ligase (lanes 3 and 6) or T4 DNA ligase (lanes 4 and 7). Lanes labeled as ‘C’ are control untreated lanes. See ‘Materials and Methods’ section for additional details.

    Techniques Used: Incubation, Plasmid Preparation, Labeling

    31) Product Images from "A novel single cell method to identify the genetic composition at a single nuclear body"

    Article Title: A novel single cell method to identify the genetic composition at a single nuclear body

    Journal: Scientific Reports

    doi: 10.1038/srep29191

    ( a ) i. Cells contained within an etched parallelogram (a “keystone”) on a coverslip are immunolabelled and Hoechst stained. ii. In the fluorescent channel of the immunolabelled signal, a Z-stack is recorded. A 2-D region of interest confined to a chosen number of stacks is targeted with two-photon irradiation. This bleaches the Hoechst in the targeted volume, causing localized DNA double strand breaks (dsbs). iii. To blunt the DNA for ligation with the blunt end probe, the cells are incubated with Klenow enzyme and DNTPs. iv. The cells are then incubated with a blunt end oligo and T4 DNA Ligase. The oligo contains priming sites that are used for the subsequent amplification steps. To prevent intra-probe ligation the oligo lacks 3′OH groups. Ligation to blunted genomic DNA occurs between a 5′phosphate contained on one strand of the oligo (binding strand) and a 3′hydroxyl of the blunted genomic DSB. ( b ) Single targeted cells (red arrowheads) are located the keystone for LMPC (Laser Micro-dissection Pressure Catapulting) isolation into lysis buffer. ( c ) The lysed cell is then subjected to PCR amplification with primers complementary to probe, to sufficient amplicon yield for sequencing. Primers used for PCR are 4bp shorter than the probe sequence so that amplicons that occur by mispriming events can be discounted by the lack of the “signature sequence” immediately following the primer sequence (see Supplementary Figure S1 ).
    Figure Legend Snippet: ( a ) i. Cells contained within an etched parallelogram (a “keystone”) on a coverslip are immunolabelled and Hoechst stained. ii. In the fluorescent channel of the immunolabelled signal, a Z-stack is recorded. A 2-D region of interest confined to a chosen number of stacks is targeted with two-photon irradiation. This bleaches the Hoechst in the targeted volume, causing localized DNA double strand breaks (dsbs). iii. To blunt the DNA for ligation with the blunt end probe, the cells are incubated with Klenow enzyme and DNTPs. iv. The cells are then incubated with a blunt end oligo and T4 DNA Ligase. The oligo contains priming sites that are used for the subsequent amplification steps. To prevent intra-probe ligation the oligo lacks 3′OH groups. Ligation to blunted genomic DNA occurs between a 5′phosphate contained on one strand of the oligo (binding strand) and a 3′hydroxyl of the blunted genomic DSB. ( b ) Single targeted cells (red arrowheads) are located the keystone for LMPC (Laser Micro-dissection Pressure Catapulting) isolation into lysis buffer. ( c ) The lysed cell is then subjected to PCR amplification with primers complementary to probe, to sufficient amplicon yield for sequencing. Primers used for PCR are 4bp shorter than the probe sequence so that amplicons that occur by mispriming events can be discounted by the lack of the “signature sequence” immediately following the primer sequence (see Supplementary Figure S1 ).

    Techniques Used: Staining, Irradiation, Ligation, Incubation, Amplification, Binding Assay, Dissection, Isolation, Lysis, Polymerase Chain Reaction, Sequencing

    32) Product Images from "Highly multiplexed simultaneous detection of RNAs and proteins in single cells"

    Article Title: Highly multiplexed simultaneous detection of RNAs and proteins in single cells

    Journal: Nature methods

    doi: 10.1038/nmeth.3742

    PLAYR enables the simultaneous quantification of specific transcripts and proteins in single cells a ) Main steps of the PLAYR protocol: 1) Fixation of cells captures their native state and permeabilization enables intracellular antibody staining and blocking of endogenous RNAses with inhibitors. 2) PLAYR probe pairs are added for proximal hybridization to target transcripts. 3) Backbone and insert oligonucleotides are added and form a circle if hybridized to PLAYR probes that are in close proximity (bound to a transcript). Insert sequences serve as cognate barcodes for targeted transcripts. 4) Backbone and insert oligonucleotides are ligated into a single-stranded DNA circle by T4 DNA ligase. 5) Rolling circle amplification of the DNA circle by phy29 polymerase. 6) Detection of rolling circle amplicons with suitably labeled oligonucleotides that bind to the insert regions. b ) Detection of transcripts for three housekeeping genes that span a wide abundance range in U937 cells by mass cytometry. c ) Quantification of CCL4 and IFNG mRNA by PLAYR and qPCR in NKL cells after stimulation with PMA-ionomycin. Measurements were performed at 4 time points in 3 replicates. d ) Simultaneous IFNG mRNA and protein quantification by mass cytometry in NKL cells at indicated time points after stimulation with PMA-ionomycin.
    Figure Legend Snippet: PLAYR enables the simultaneous quantification of specific transcripts and proteins in single cells a ) Main steps of the PLAYR protocol: 1) Fixation of cells captures their native state and permeabilization enables intracellular antibody staining and blocking of endogenous RNAses with inhibitors. 2) PLAYR probe pairs are added for proximal hybridization to target transcripts. 3) Backbone and insert oligonucleotides are added and form a circle if hybridized to PLAYR probes that are in close proximity (bound to a transcript). Insert sequences serve as cognate barcodes for targeted transcripts. 4) Backbone and insert oligonucleotides are ligated into a single-stranded DNA circle by T4 DNA ligase. 5) Rolling circle amplification of the DNA circle by phy29 polymerase. 6) Detection of rolling circle amplicons with suitably labeled oligonucleotides that bind to the insert regions. b ) Detection of transcripts for three housekeeping genes that span a wide abundance range in U937 cells by mass cytometry. c ) Quantification of CCL4 and IFNG mRNA by PLAYR and qPCR in NKL cells after stimulation with PMA-ionomycin. Measurements were performed at 4 time points in 3 replicates. d ) Simultaneous IFNG mRNA and protein quantification by mass cytometry in NKL cells at indicated time points after stimulation with PMA-ionomycin.

    Techniques Used: Staining, Blocking Assay, Hybridization, Amplification, Labeling, Mass Cytometry, Real-time Polymerase Chain Reaction

    33) Product Images from "Assessing the Amount of Quadruplex Structures Present within G2-Tract Synthetic Random-Sequence DNA Libraries"

    Article Title: Assessing the Amount of Quadruplex Structures Present within G2-Tract Synthetic Random-Sequence DNA Libraries

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064131

    Scheme for the ligation of the representative sequence DGR36 to PCR adapters. (A) DGR36 (red) was ligated to 5′ (black) and 3′ (blue) adapters, with two template oligonucleotides (grey). The asterisk represents a 32 P labelled phosphate. (B) Ligation of one and two adapters to DGR36. The 3′ adapter was labelled with 32 P (lane 1) and incubated with T4 DNA ligase, DGR36 and fully complementary template (lane 2), as well as all these oligonucleotides plus a 5′ adapter and fully complementary template (lane 3). The same reactions were performed with the fully complementary templates replaced with partially randomized 3′ templates (lane 4) and both 3′ and 5′ templates (lane 5). The p and App on the 3′ adapters in lanes 4 and 5 represent 5′ phosphorylated and 5′ adenylated adapters, respectively.
    Figure Legend Snippet: Scheme for the ligation of the representative sequence DGR36 to PCR adapters. (A) DGR36 (red) was ligated to 5′ (black) and 3′ (blue) adapters, with two template oligonucleotides (grey). The asterisk represents a 32 P labelled phosphate. (B) Ligation of one and two adapters to DGR36. The 3′ adapter was labelled with 32 P (lane 1) and incubated with T4 DNA ligase, DGR36 and fully complementary template (lane 2), as well as all these oligonucleotides plus a 5′ adapter and fully complementary template (lane 3). The same reactions were performed with the fully complementary templates replaced with partially randomized 3′ templates (lane 4) and both 3′ and 5′ templates (lane 5). The p and App on the 3′ adapters in lanes 4 and 5 represent 5′ phosphorylated and 5′ adenylated adapters, respectively.

    Techniques Used: Ligation, Sequencing, Polymerase Chain Reaction, Incubation

    34) Product Images from "Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain"

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039251

    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.
    Figure Legend Snippet: 15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    12% denaturing PAGE for the ligation products of linkers A–B treated with CIAP. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.
    Figure Legend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B treated with CIAP. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Positive Control, Negative Control

    12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.
    Figure Legend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.
    Figure Legend Snippet: The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Electrophoresis, Negative Control, Positive Control

    35) Product Images from "Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain"

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039251

    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.
    Figure Legend Snippet: 15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    12% denaturing PAGE for the ligation products of linkers A–B treated with CIAP. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.
    Figure Legend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B treated with CIAP. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Positive Control, Negative Control

    12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.
    Figure Legend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.
    Figure Legend Snippet: The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Electrophoresis, Negative Control, Positive Control

    36) Product Images from "Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain"

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039251

    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.
    Figure Legend Snippet: 15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    12% denaturing PAGE for the ligation products of linkers A–B treated with CIAP. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.
    Figure Legend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B treated with CIAP. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Positive Control, Negative Control

    12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.
    Figure Legend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.
    Figure Legend Snippet: The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Electrophoresis, Negative Control, Positive Control

    37) Product Images from "Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain"

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039251

    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.
    Figure Legend Snippet: 15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    12% denaturing PAGE for the ligation products of linkers A–B treated with CIAP. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.
    Figure Legend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B treated with CIAP. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Positive Control, Negative Control

    12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.
    Figure Legend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.
    Figure Legend Snippet: The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Electrophoresis, Negative Control, Positive Control

    38) Product Images from "A systematic, ligation-based approach to study RNA modifications"

    Article Title: A systematic, ligation-based approach to study RNA modifications

    Journal: RNA

    doi: 10.1261/rna.208906

    Outline of the ligation method to study RNA modifications. ( A ) The RNA (black line) containing an unmodified or modified nucleotide (open or filled black circles) is hybridized with two oligonucleotides. These oligonucleotides are ligated by the T4 DNA ligase using this RNA as a template (triangle). The D- and N-oligos have different nucleotide compositions (green or red circles) at the ligation junction or different ligation sites. ( B ) For the D-oligo (green line), ligation proceeds very efficiently when the RNA is unmodified, but very poorly when the RNA is modified (or the other way around). For the N-oligo (red line), ligation has a constant yield regardless of the status of RNA modification. The amount of D-oligo ligation product corresponds to the amount of unmodified over modified RNA template (or modified over unmodified). The amount of N-oligo product corresponds to the sum of the unmodified and modified RNA.
    Figure Legend Snippet: Outline of the ligation method to study RNA modifications. ( A ) The RNA (black line) containing an unmodified or modified nucleotide (open or filled black circles) is hybridized with two oligonucleotides. These oligonucleotides are ligated by the T4 DNA ligase using this RNA as a template (triangle). The D- and N-oligos have different nucleotide compositions (green or red circles) at the ligation junction or different ligation sites. ( B ) For the D-oligo (green line), ligation proceeds very efficiently when the RNA is unmodified, but very poorly when the RNA is modified (or the other way around). For the N-oligo (red line), ligation has a constant yield regardless of the status of RNA modification. The amount of D-oligo ligation product corresponds to the amount of unmodified over modified RNA template (or modified over unmodified). The amount of N-oligo product corresponds to the sum of the unmodified and modified RNA.

    Techniques Used: Ligation, Modification

    39) Product Images from "Practical Synthesis of Cap‐4 RNA"

    Article Title: Practical Synthesis of Cap‐4 RNA

    Journal: Chembiochem

    doi: 10.1002/cbic.201900590

    Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using T4 DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μ m RNA 10 , 12.5 μ m RNA 11 , 12.5 μ m splint; 0.5 m m ATP, 40 m m Tris ⋅ HCl (pH 7.8), 10 m m MgCl 2 , 10 m m DTT, 5 % ( w / v ) PEG 4000, 0.5 U μL −1 T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.
    Figure Legend Snippet: Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using T4 DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μ m RNA 10 , 12.5 μ m RNA 11 , 12.5 μ m splint; 0.5 m m ATP, 40 m m Tris ⋅ HCl (pH 7.8), 10 m m MgCl 2 , 10 m m DTT, 5 % ( w / v ) PEG 4000, 0.5 U μL −1 T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.

    Techniques Used: Ligation, Sequencing, High Performance Liquid Chromatography, Purification

    40) Product Images from "Periplasmic Expression of TNF Related Apoptosis Inducing Ligand (TRAIL) in E.coli"

    Article Title: Periplasmic Expression of TNF Related Apoptosis Inducing Ligand (TRAIL) in E.coli

    Journal: Iranian Journal of Pharmaceutical Research : IJPR

    doi:

    Cloning of OmpA-TRAIL fragment in pET-22b expression plasmid. OmpA-TRAIL fragment and pET-22b plasmid were digested separately with restriction Enzymes Nde I and Xho I. Then, digested fragment and plasmid were ligated using T4 DNA ligase.
    Figure Legend Snippet: Cloning of OmpA-TRAIL fragment in pET-22b expression plasmid. OmpA-TRAIL fragment and pET-22b plasmid were digested separately with restriction Enzymes Nde I and Xho I. Then, digested fragment and plasmid were ligated using T4 DNA ligase.

    Techniques Used: Clone Assay, Positron Emission Tomography, Expressing, Plasmid Preparation

    Related Articles

    Nucleic Acid Electrophoresis:

    Article Title: Reverse transcriptase and endonuclease activities encoded by Penelope-like retroelements
    Article Snippet: .. After addition of 0.5 Weiss unit of T4 DNA ligase (Fermentas, Hanover, MD), reactions were incubated at 16°C and analyzed by denaturing gel electrophoresis as above. ..

    Synthesized:

    Article Title: Practical Synthesis of Cap‐4 RNA
    Article Snippet: .. The 38‐nt T. cruzi cap‐4 RNA was prepared by splinted enzymatic ligation of an 11‐nt cap‐4 RNA and a chemically synthesized 5′‐phosphorylated 27‐nt RNA by using T4 DNA ligase (Thermo Fisher) in analogy to ref. . ..

    Ligation:

    Article Title: Identification of recognition residues for ligation-based detection and quantitation of pseudouridine and N6-methyladenosine
    Article Snippet: .. The optimized ligation reactions were carried out with 0.15 µM 30-mer RNA with or without Ψ or m6 A modifications, 0.5 µM floater and 0.38 µM of 5′-32 P-labeled anchor in 66 mM Tris–HCl, pH 7.6, 0.5 mM ZnCl2 , 10 mM DTT, 66 µM ATP, 15% DMSO and 0.25 U/µl T4 DNA ligase (USB Inc.). .. All components were mixed and incubated at 16°C for 16 h, and the ligation products separated on denaturing polyacrylamide gels containing 7 M urea.

    Article Title: Practical Synthesis of Cap‐4 RNA
    Article Snippet: .. The 38‐nt T. cruzi cap‐4 RNA was prepared by splinted enzymatic ligation of an 11‐nt cap‐4 RNA and a chemically synthesized 5′‐phosphorylated 27‐nt RNA by using T4 DNA ligase (Thermo Fisher) in analogy to ref. . ..

    Article Title: Profiling non-lysyl tRNAs in HIV-1
    Article Snippet: .. The ligation reaction was carried out overnight (∼16 h) at 16°C with 0.13 μg/μL total RNA in 1X T4 DNA ligase buffer, 0.5 U/μL T4 DNA ligase (USB Corporation, 70042X), 15% DMSO, and 4.5 μM labeling oligonucleotide. .. Hybridization was performed at 60°C overnight (∼16 h) with 1–2 μg each of Cy3- or Cy5-labeled total RNA as previously described ( ).

    Labeling:

    Article Title: Profiling non-lysyl tRNAs in HIV-1
    Article Snippet: .. The ligation reaction was carried out overnight (∼16 h) at 16°C with 0.13 μg/μL total RNA in 1X T4 DNA ligase buffer, 0.5 U/μL T4 DNA ligase (USB Corporation, 70042X), 15% DMSO, and 4.5 μM labeling oligonucleotide. .. Hybridization was performed at 60°C overnight (∼16 h) with 1–2 μg each of Cy3- or Cy5-labeled total RNA as previously described ( ).

    Purification:

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis
    Article Snippet: .. First, the purified product of PCR for the FSH1 gene was ligated into pUC-PUT following DNA digestion with Xho I and Hin dIII, and ligated by T4 DNA ligase (Invitrogen; Thermo Fisher Scientific, Inc.). ..

    Incubation:

    Article Title: Autophosphorylation-dependent remodeling of the DNA-dependent protein kinase catalytic subunit regulates ligation of DNA ends
    Article Snippet: .. Reactions were started by the addition of 0.5 U of T4 DNA ligase (Invitrogen, Carlsbad, CA) and incubated at 37°C. ..

    Article Title: Reverse transcriptase and endonuclease activities encoded by Penelope-like retroelements
    Article Snippet: .. After addition of 0.5 Weiss unit of T4 DNA ligase (Fermentas, Hanover, MD), reactions were incubated at 16°C and analyzed by denaturing gel electrophoresis as above. ..

    Polymerase Chain Reaction:

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis
    Article Snippet: .. First, the purified product of PCR for the FSH1 gene was ligated into pUC-PUT following DNA digestion with Xho I and Hin dIII, and ligated by T4 DNA ligase (Invitrogen; Thermo Fisher Scientific, Inc.). ..

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    • t4 dna ligase  (Thermo Fisher)
    99
    Thermo Fisher t4 dna ligase
    Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using <t>T4</t> DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μ m RNA 10 , 12.5 μ m RNA 11 , 12.5 μ m splint; 0.5 m m ATP, 40 m m Tris ⋅ HCl (pH 7.8), 10 m m MgCl 2 , 10 m m DTT, 5 % ( w / v ) PEG 4000, 0.5 U μL −1 T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 550 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase/product/Thermo Fisher
    Average 99 stars, based on 550 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2020-07
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    t4 dna ligase buffer  (Thermo Fisher)
    96
    Thermo Fisher t4 dna ligase buffer
    Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard <t>T4</t> DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.
    T4 Dna Ligase Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase buffer/product/Thermo Fisher
    Average 96 stars, based on 96 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase buffer - by Bioz Stars, 2020-07
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    Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using T4 DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μ m RNA 10 , 12.5 μ m RNA 11 , 12.5 μ m splint; 0.5 m m ATP, 40 m m Tris ⋅ HCl (pH 7.8), 10 m m MgCl 2 , 10 m m DTT, 5 % ( w / v ) PEG 4000, 0.5 U μL −1 T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.

    Journal: Chembiochem

    Article Title: Practical Synthesis of Cap‐4 RNA

    doi: 10.1002/cbic.201900590

    Figure Lengend Snippet: Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using T4 DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μ m RNA 10 , 12.5 μ m RNA 11 , 12.5 μ m splint; 0.5 m m ATP, 40 m m Tris ⋅ HCl (pH 7.8), 10 m m MgCl 2 , 10 m m DTT, 5 % ( w / v ) PEG 4000, 0.5 U μL −1 T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.

    Article Snippet: The 38‐nt T. cruzi cap‐4 RNA was prepared by splinted enzymatic ligation of an 11‐nt cap‐4 RNA and a chemically synthesized 5′‐phosphorylated 27‐nt RNA by using T4 DNA ligase (Thermo Fisher) in analogy to ref. .

    Techniques: Ligation, Sequencing, High Performance Liquid Chromatography, Purification

    Single-stranded DNA ligation with T4 DNA ligase and CircLigase. A pool of 60 nt acceptor oligonucleotides (‘60N’) were ligated to 10 pmol of a 3΄ biotinylated donor oligonucleotide (CL78) using either T4 DNA ligase in the presence of a splinter oligonucleotide (TL38) or CircLigase. Ligation products were visualized on a 10% denaturing polyacrylamide gel stained with SybrGold. Band shifts from 60 nt to 80 nt indicate successful ligation. Schematic overviews of the reaction schemes are shown on top. The scheme developed by Kwok et al . ( 19 ) is shown for comparison. M: Single-stranded DNA size marker.

    Journal: Nucleic Acids Research

    Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

    doi: 10.1093/nar/gkx033

    Figure Lengend Snippet: Single-stranded DNA ligation with T4 DNA ligase and CircLigase. A pool of 60 nt acceptor oligonucleotides (‘60N’) were ligated to 10 pmol of a 3΄ biotinylated donor oligonucleotide (CL78) using either T4 DNA ligase in the presence of a splinter oligonucleotide (TL38) or CircLigase. Ligation products were visualized on a 10% denaturing polyacrylamide gel stained with SybrGold. Band shifts from 60 nt to 80 nt indicate successful ligation. Schematic overviews of the reaction schemes are shown on top. The scheme developed by Kwok et al . ( 19 ) is shown for comparison. M: Single-stranded DNA size marker.

    Article Snippet: Splinted end-to-end ligation of single-stranded DNA using T4 DNA ligase To explore the efficiency of splinted end-to-end ligation of single stranded DNA with T4 DNA ligase in the absence of hair-pin structures, we designed a ligation scheme where the splinter oligonucleotide is hybridized to a biotinylated adapter oligonucleotide (the donor), allowing for subsequent immobilization of ligation products on beads and removal of the splinter by mild heat treatment.

    Techniques: DNA Ligation, Ligation, Staining, Marker

    Library preparation methods for highly degraded DNA. ( A ) In the single-stranded library preparation method described here (ssDNA2.0), DNA fragments (black) are 5΄ and 3΄ dephosphorylated and separated into single strands by heat denaturation. 3΄ biotinylated adapter molecules (red) are attached to the 3΄ ends of the DNA fragments via hybridization to a stretch of six random nucleotides (marked as ‘N’) belonging to a splinter oligonucleotide complementary to the adapter and nick closure with T4 DNA ligase. Following the immobilization of the ligation products on streptavidin-coated beads, the splinter oligonucleotide is removed by bead wash at an elevated temperature. Synthesis of the second strand is carried out using the Klenow fragment of Escherichia coli DNA polymerase I and a primer with phosphorothioate backbone modifications (red stars) to prevent exonucleolytic degradation. Unincorporated primers are removed through a bead wash at an elevated temperature, preventing the formation of adapter dimers in the subsequent blunt-end ligation reaction, which is again catalyzed by T4 DNA ligase. Adapter self-ligation is prevented through a 3΄ dideoxy modification in the adapter. The final library strand is released from the beads by heat denaturation. ( B ) In the single-stranded library preparation method originally described in Gansauge and Meyer, ( 4 ), the first adapter was attached through true single-stranded DNA ligation using CircLigase. The large fragment of Bst DNA polymerase was used to copy the template strand, leaving overhanging 3΄ nucleotides, which had to be removed in a blunt-end repair reaction using T4 DNA polymerase. ( C ) The ‘454’ method of double-stranded library preparation in the implementation of Meyer and Kircher, ( 23 ), is based on non-directional blunt-end ligation of a mixture of two adapters to blunt-end repaired DNA fragments using T4 DNA ligase. To prevent adapter self-ligation, no phosphate groups are present at the 5΄ ends of the adapters, resulting in the ligation of the adapter strands only and necessitating subsequent nick fill-in with a strand-displacing polymerase. Intermittent DNA purification steps are required in-between enzymatic reactions. ( D ) The ‘Illumina’ method of double-stranded library preparation, shown here as implemented in New England Biolabs’ NEBNext Ultra II kit, requires the addition of A-overhangs (marked as ‘A’) to blunt-end repaired DNA fragments using a 3΄-5΄ exonuclease deletion mutant of the Klenow fragment of E. coli DNA polymerase I. Both adapter sequences are combined into one bell-shaped structure, which carries a 3΄ T overhang to allow sticky end ligation with T4 DNA ligase. Following ligation, adapter strands are separated by excision of uracil. Excess adapters and adapter dimers are removed through size-selective purification.

    Journal: Nucleic Acids Research

    Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

    doi: 10.1093/nar/gkx033

    Figure Lengend Snippet: Library preparation methods for highly degraded DNA. ( A ) In the single-stranded library preparation method described here (ssDNA2.0), DNA fragments (black) are 5΄ and 3΄ dephosphorylated and separated into single strands by heat denaturation. 3΄ biotinylated adapter molecules (red) are attached to the 3΄ ends of the DNA fragments via hybridization to a stretch of six random nucleotides (marked as ‘N’) belonging to a splinter oligonucleotide complementary to the adapter and nick closure with T4 DNA ligase. Following the immobilization of the ligation products on streptavidin-coated beads, the splinter oligonucleotide is removed by bead wash at an elevated temperature. Synthesis of the second strand is carried out using the Klenow fragment of Escherichia coli DNA polymerase I and a primer with phosphorothioate backbone modifications (red stars) to prevent exonucleolytic degradation. Unincorporated primers are removed through a bead wash at an elevated temperature, preventing the formation of adapter dimers in the subsequent blunt-end ligation reaction, which is again catalyzed by T4 DNA ligase. Adapter self-ligation is prevented through a 3΄ dideoxy modification in the adapter. The final library strand is released from the beads by heat denaturation. ( B ) In the single-stranded library preparation method originally described in Gansauge and Meyer, ( 4 ), the first adapter was attached through true single-stranded DNA ligation using CircLigase. The large fragment of Bst DNA polymerase was used to copy the template strand, leaving overhanging 3΄ nucleotides, which had to be removed in a blunt-end repair reaction using T4 DNA polymerase. ( C ) The ‘454’ method of double-stranded library preparation in the implementation of Meyer and Kircher, ( 23 ), is based on non-directional blunt-end ligation of a mixture of two adapters to blunt-end repaired DNA fragments using T4 DNA ligase. To prevent adapter self-ligation, no phosphate groups are present at the 5΄ ends of the adapters, resulting in the ligation of the adapter strands only and necessitating subsequent nick fill-in with a strand-displacing polymerase. Intermittent DNA purification steps are required in-between enzymatic reactions. ( D ) The ‘Illumina’ method of double-stranded library preparation, shown here as implemented in New England Biolabs’ NEBNext Ultra II kit, requires the addition of A-overhangs (marked as ‘A’) to blunt-end repaired DNA fragments using a 3΄-5΄ exonuclease deletion mutant of the Klenow fragment of E. coli DNA polymerase I. Both adapter sequences are combined into one bell-shaped structure, which carries a 3΄ T overhang to allow sticky end ligation with T4 DNA ligase. Following ligation, adapter strands are separated by excision of uracil. Excess adapters and adapter dimers are removed through size-selective purification.

    Article Snippet: Splinted end-to-end ligation of single-stranded DNA using T4 DNA ligase To explore the efficiency of splinted end-to-end ligation of single stranded DNA with T4 DNA ligase in the absence of hair-pin structures, we designed a ligation scheme where the splinter oligonucleotide is hybridized to a biotinylated adapter oligonucleotide (the donor), allowing for subsequent immobilization of ligation products on beads and removal of the splinter by mild heat treatment.

    Techniques: Hybridization, Ligation, Modification, DNA Ligation, DNA Purification, Mutagenesis, Purification

    Effects of single-stranded ligation schemes on library characteristics. ( A ) Informative sequence content of libraries prepared with CircLigase and T4 DNA ligase as a function of the input volume of ancient DNA extract used for library preparation. ( B ) Average GC content of the sequences obtained with the two ligation schemes. Note that the average GC content exceeds that of a typical mammalian genome because most sequences derive from microbial DNA, which is the dominant source of DNA in most ancient bones. ( C ) Fragment size distribution in the libraries as inferred from overlap-merged paired-end reads. Short artifacts in the library prepared from extremely little input DNA (corresponding to ∼1 mg bone) are mainly due to the incorporation of splinter fragments. ( D ) Frequencies of damage-induced C to T substitutions near the 5΄ and 3΄ ends of sequences.

    Journal: Nucleic Acids Research

    Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

    doi: 10.1093/nar/gkx033

    Figure Lengend Snippet: Effects of single-stranded ligation schemes on library characteristics. ( A ) Informative sequence content of libraries prepared with CircLigase and T4 DNA ligase as a function of the input volume of ancient DNA extract used for library preparation. ( B ) Average GC content of the sequences obtained with the two ligation schemes. Note that the average GC content exceeds that of a typical mammalian genome because most sequences derive from microbial DNA, which is the dominant source of DNA in most ancient bones. ( C ) Fragment size distribution in the libraries as inferred from overlap-merged paired-end reads. Short artifacts in the library prepared from extremely little input DNA (corresponding to ∼1 mg bone) are mainly due to the incorporation of splinter fragments. ( D ) Frequencies of damage-induced C to T substitutions near the 5΄ and 3΄ ends of sequences.

    Article Snippet: Splinted end-to-end ligation of single-stranded DNA using T4 DNA ligase To explore the efficiency of splinted end-to-end ligation of single stranded DNA with T4 DNA ligase in the absence of hair-pin structures, we designed a ligation scheme where the splinter oligonucleotide is hybridized to a biotinylated adapter oligonucleotide (the donor), allowing for subsequent immobilization of ligation products on beads and removal of the splinter by mild heat treatment.

    Techniques: Ligation, Sequencing, Ancient DNA Assay

    DNA barcoding experimental scheme. Target DNA strands are immobilized on a microscope slide, and dye-labeled barcodes are introduced together with T4 DNA ligase in the microfluidic chamber (1). Complementary barcodes bind transiently to the target site (2), whereas mismatched barcodes bind on an even shorter timescale (2′). Successful ligation is observed for the complementary barcodes (3) but not for the mismatched barcodes (3′). Ligation product shows stable binding to the target DNA (4), whereas mismatched barcodes dissociate and are washed away before imaging. To see this figure in color, go online.

    Journal: Biophysical Journal

    Article Title: Multiplex Single-Molecule DNA Barcoding Using an Oligonucleotide Ligation Assay

    doi: 10.1016/j.bpj.2018.08.013

    Figure Lengend Snippet: DNA barcoding experimental scheme. Target DNA strands are immobilized on a microscope slide, and dye-labeled barcodes are introduced together with T4 DNA ligase in the microfluidic chamber (1). Complementary barcodes bind transiently to the target site (2), whereas mismatched barcodes bind on an even shorter timescale (2′). Successful ligation is observed for the complementary barcodes (3) but not for the mismatched barcodes (3′). Ligation product shows stable binding to the target DNA (4), whereas mismatched barcodes dissociate and are washed away before imaging. To see this figure in color, go online.

    Article Snippet: For ligation, T4 DNA ligase was used in standard conditions (25°C, 10 mM MgCl2 ), and the GC content of the target site was ∼50%.

    Techniques: Microscopy, Labeling, Ligation, Binding Assay, Imaging

    Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard T4 DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.

    Journal: Nucleic Acids Research

    Article Title: SPlinted Ligation Adapter Tagging (SPLAT), a novel library preparation method for whole genome bisulphite sequencing

    doi: 10.1093/nar/gkw1110

    Figure Lengend Snippet: Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard T4 DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.

    Article Snippet: For the 3΄-end ligation; adapter ss1 (final conc 10 μM), T4 DNA ligase buffer (40 mM Tris–HCl pH 7.8,10 mM MgCl2 , 10 mM DTT, 0.5 mM ATP), PEG4000 (5% w/v) and 30 units T4 DNA ligase (Thermo Fisher Scientific) and nuclease free water was added to the sample on ice, in a total volume of 30 μl.

    Techniques: Bisulfite Sequencing, Methylation, Construct, Amplification, Polymerase Chain Reaction, Next-Generation Sequencing, Sequencing, DNA Methylation Assay, Random Hexamer Labeling, DNA Ligation, Modification, Ligation