t4 dna ligase  (Thermo Fisher)


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    Name:
    T4 DNA Ligase (5 U/µL)
    Description:
    T4 DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of ATP between double-stranded DNAs with 3´ hydroxyl and 5´ phosphate termini. The unique T4 DNA Ligase buffer optimizes ligation, which can be performed in 5 minutes (1). Single-stranded nucleic acids are not substrates for this enzyme. A T4 DNA Ligase Technical Bulletin is available.Applications: Cloning (blunt-end or cohesive-end ligation) (2). Adding linkers or adapters to blunt-ended DNA (2).Source: Purified from E. coli œ lysogen NM989.Performance and Quality Testing: Endodeoxyribonuclease, 3´ and 5´ exodeoxyribonuclease assays; ligation efficiency tested.Unit Definition: One unit catalyzes the exchange of 1 nmol 32P-labeled pyrophosphate into ATP in 20 min. at 37°C. (One unit is equal to approximately 300 cohesive-end ligation units.)Unit Reaction Conditions: 66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl2 , 10 mM DTT, 66 µM ATP, 3.3 µM 32 P-labeled pyrophosphate, and enzyme in 0.1 ml for 20 min. at 37°C.
    Catalog Number:
    15224041
    Price:
    None
    Applications:
    ChIP-on-Chip|Cloning|RNAi, Epigenetics & Non-Coding RNA Research|Chromatin Biology|Restriction Enzyme Cloning
    Size:
    250 units
    Category:
    Proteins, Enzymes, & Peptides, PCR & Cloning Enzymes, DNA⁄RNA Modifying Enzymes
    Score:
    85
    		Buy from Supplier

    Structured Review

    Thermo Fisher t4 dna ligase
    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).
    T4 DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of ATP between double-stranded DNAs with 3´ hydroxyl and 5´ phosphate termini. The unique T4 DNA Ligase buffer optimizes ligation, which can be performed in 5 minutes (1). Single-stranded nucleic acids are not substrates for this enzyme. A T4 DNA Ligase Technical Bulletin is available.Applications: Cloning (blunt-end or cohesive-end ligation) (2). Adding linkers or adapters to blunt-ended DNA (2).Source: Purified from E. coli œ lysogen NM989.Performance and Quality Testing: Endodeoxyribonuclease, 3´ and 5´ exodeoxyribonuclease assays; ligation efficiency tested.Unit Definition: One unit catalyzes the exchange of 1 nmol 32P-labeled pyrophosphate into ATP in 20 min. at 37°C. (One unit is equal to approximately 300 cohesive-end ligation units.)Unit Reaction Conditions: 66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl2 , 10 mM DTT, 66 µM ATP, 3.3 µM 32 P-labeled pyrophosphate, and enzyme in 0.1 ml for 20 min. at 37°C.
    https://www.bioz.com/result/t4 dna ligase/product/Thermo Fisher
    Average 99 stars, based on 0 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2019-10
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    Images

    1) 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

    2) 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

    3) 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

    4) 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

    5) Product Images from "An In Vitro DNA Double-Strand Break Repair Assay Based on End-Joining of Defined Duplex Oligonucleotides"

    Article Title: An In Vitro DNA Double-Strand Break Repair Assay Based on End-Joining of Defined Duplex Oligonucleotides

    Journal:

    doi: 10.1007/978-1-61779-998-3_33

    A simple schematic representation of the basic assay plan and an example of a typical set of results obtained for direct ligation with T4 DNA ligase (positive control) of an undamaged control oligo substrate, vs. end-joining of the same substrate by HeLa
    Figure Legend Snippet: A simple schematic representation of the basic assay plan and an example of a typical set of results obtained for direct ligation with T4 DNA ligase (positive control) of an undamaged control oligo substrate, vs. end-joining of the same substrate by HeLa

    Techniques Used: Ligation, Positive Control

    6) 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, Gas Chromatography

    7) Product Images from "The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly"

    Article Title: The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt122

    In vitro seamless assembly of the whole actinorhodin biosynthetic cluster from multiple restriction fragments. ( A ) The schematic diagram of the PCR amplicons. The 29-kb actinorhodin biosynthetic cluster was divided into four fragments, which were then PCR amplified with primers specified ( Supplementary Table S1 ). ( B ) Errorless demethylated fragments I (7420 bp), II (8171 bp), III (6410 bp) and IV (6686 bp) were released from pBluescript II KS by XbaI digestion, which were then ligated with a designed adaptor klf.ML2 ( Figure 1 B and Supplementary Table S1 ) and digested with MspJI as described in the ‘Materials and Methods’ section. The MspJI-treated fragments were used for ligation with T4 DNA ligase at 16°C for 2 h. ( C ) Fragments I–II and III–IV were ligated with pHI with (‘L+’) or without (‘L−’) the addition of T4 DNA ligase. The synthesized DNA could be viewed in the ‘L+’ lane together with the disappearance of the substrate fragments. The ligation reaction was performed at temperature 22°C for 8 h. ( D ) The BamHI restriction map of the synthesized plasmid pHIW. The theoretical restriction fragments of pHIW contains fragments of 11 848, 7001, 5804, 3923, 2172, 1731, 1191, 1029, 928 and 743 bp in size. The 743-bp fragment was run out of the gel and was not shown. ( E ) Heterologous expression of the assembled actinorhodin biosynthetic cluster in Streptomyces strain 4F (4F/pHIW), using 4F integrated plasmid pHI (4F/pHI) as a negative control. Strains were cultured on R2YE medium at 30°C for 2 days.
    Figure Legend Snippet: In vitro seamless assembly of the whole actinorhodin biosynthetic cluster from multiple restriction fragments. ( A ) The schematic diagram of the PCR amplicons. The 29-kb actinorhodin biosynthetic cluster was divided into four fragments, which were then PCR amplified with primers specified ( Supplementary Table S1 ). ( B ) Errorless demethylated fragments I (7420 bp), II (8171 bp), III (6410 bp) and IV (6686 bp) were released from pBluescript II KS by XbaI digestion, which were then ligated with a designed adaptor klf.ML2 ( Figure 1 B and Supplementary Table S1 ) and digested with MspJI as described in the ‘Materials and Methods’ section. The MspJI-treated fragments were used for ligation with T4 DNA ligase at 16°C for 2 h. ( C ) Fragments I–II and III–IV were ligated with pHI with (‘L+’) or without (‘L−’) the addition of T4 DNA ligase. The synthesized DNA could be viewed in the ‘L+’ lane together with the disappearance of the substrate fragments. The ligation reaction was performed at temperature 22°C for 8 h. ( D ) The BamHI restriction map of the synthesized plasmid pHIW. The theoretical restriction fragments of pHIW contains fragments of 11 848, 7001, 5804, 3923, 2172, 1731, 1191, 1029, 928 and 743 bp in size. The 743-bp fragment was run out of the gel and was not shown. ( E ) Heterologous expression of the assembled actinorhodin biosynthetic cluster in Streptomyces strain 4F (4F/pHIW), using 4F integrated plasmid pHI (4F/pHI) as a negative control. Strains were cultured on R2YE medium at 30°C for 2 days.

    Techniques Used: In Vitro, Polymerase Chain Reaction, Amplification, Ligation, Synthesized, Plasmid Preparation, Expressing, Negative Control, Cell Culture

    8) 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, Gas Chromatography

    9) 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, Gas Chromatography

    10) 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

    11) Product Images from "Highly efficient preparation of single-stranded DNA rings by T4 ligase at abnormally low Mg(II) concentration"

    Article Title: Highly efficient preparation of single-stranded DNA rings by T4 ligase at abnormally low Mg(II) concentration

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx553

    Cyclization of l-DNA 66 by T4 DNA ligase. Schematic views of the formation of ( A ) single-stranded DNA ring (c-DNA 66 ) with the assistance of splint-12 nt and ( B ) polymers from multiple l-DNA 66 strands. The single-stranded DNA substrate (l-DNA 66 ) bears a phosphate at the 5′-terminus. ( C ) Effects of [l-DNA 66 ] 0 on the formation of c-DNA 66 and the polymers in 1× T4 ligase buffer (conventional method). The selectivity for the formation of c-DNA 66 is presented below the corresponding band. [l-DNA 66 ] 0 /[splint-12 nt] 0 = 1/2 at 20°C for 12 h. All the DNA substrate was added to the mixture all at once at the beginning of the reaction.
    Figure Legend Snippet: Cyclization of l-DNA 66 by T4 DNA ligase. Schematic views of the formation of ( A ) single-stranded DNA ring (c-DNA 66 ) with the assistance of splint-12 nt and ( B ) polymers from multiple l-DNA 66 strands. The single-stranded DNA substrate (l-DNA 66 ) bears a phosphate at the 5′-terminus. ( C ) Effects of [l-DNA 66 ] 0 on the formation of c-DNA 66 and the polymers in 1× T4 ligase buffer (conventional method). The selectivity for the formation of c-DNA 66 is presented below the corresponding band. [l-DNA 66 ] 0 /[splint-12 nt] 0 = 1/2 at 20°C for 12 h. All the DNA substrate was added to the mixture all at once at the beginning of the reaction.

    Techniques Used:

    Effects of ( A ) [Mg 2+ ] 0 and ( B ) [ATP] 0 on the cyclization of l-DNA 66 by T4 DNA ligase. [l-DNA 66 ] 0 = 1 μM, [splint-12 nt] 0 = 2 μM, 5 U T4 DNA ligase at 20°C and 12 h. [ATP] 0 = 25 μM, [DTT] = 0.5 mM, and [Tris–HCl] = 2 mM in ( A ), whereas [MgCl 2 ] = 0.5 mM, [DTT] = 0.5 mM, and [Tris–HCl] = 2 mM in ( B ).
    Figure Legend Snippet: Effects of ( A ) [Mg 2+ ] 0 and ( B ) [ATP] 0 on the cyclization of l-DNA 66 by T4 DNA ligase. [l-DNA 66 ] 0 = 1 μM, [splint-12 nt] 0 = 2 μM, 5 U T4 DNA ligase at 20°C and 12 h. [ATP] 0 = 25 μM, [DTT] = 0.5 mM, and [Tris–HCl] = 2 mM in ( A ), whereas [MgCl 2 ] = 0.5 mM, [DTT] = 0.5 mM, and [Tris–HCl] = 2 mM in ( B ).

    Techniques Used:

    Effects of the concentration of T4 ligase buffer on the efficiencies of cyclization of l-DNA 66 and its polymerization. All the DNA substrate was added to the mixture all at once at the beginning of the reaction. The selectivity for the formation of c-DNA 66 is presented below the corresponding band. The reaction conditions: [l-DNA 66 ] 0 = 1 μM; [splint-12 nt] 0 = 2 μM; 5 U T4 DNA ligase at 20°C and 12 h. Note that 1 × T4 ligase buffer contains 10 mM MgCl 2 , 500 μM ATP, 10 mM DTT and 40 mM Tris-HCl.
    Figure Legend Snippet: Effects of the concentration of T4 ligase buffer on the efficiencies of cyclization of l-DNA 66 and its polymerization. All the DNA substrate was added to the mixture all at once at the beginning of the reaction. The selectivity for the formation of c-DNA 66 is presented below the corresponding band. The reaction conditions: [l-DNA 66 ] 0 = 1 μM; [splint-12 nt] 0 = 2 μM; 5 U T4 DNA ligase at 20°C and 12 h. Note that 1 × T4 ligase buffer contains 10 mM MgCl 2 , 500 μM ATP, 10 mM DTT and 40 mM Tris-HCl.

    Techniques Used: Concentration Assay

    Effects of the length of splints on the efficiencies of cyclization of l-DNA 66 and its polymerization under conventional conditions. All the DNA substrate was added to the mixture all at once at the beginning of the reaction. Each of the splints is complementary to equal number of nucleotides in the 5′- and 3′-ends of l-DNA 66 , respectively (the binding mode of splint-12 nt is presented in Figure 1A ). The reaction conditions: [l-DNA 66 ] 0 = 5 μM; [splint] 0 = 10 μM; 20 U T4 DNA ligase in 1 × T4 ligase buffer at 20°C and 12 h. The sequences of splints were listed in Supplementary Table S1 .
    Figure Legend Snippet: Effects of the length of splints on the efficiencies of cyclization of l-DNA 66 and its polymerization under conventional conditions. All the DNA substrate was added to the mixture all at once at the beginning of the reaction. Each of the splints is complementary to equal number of nucleotides in the 5′- and 3′-ends of l-DNA 66 , respectively (the binding mode of splint-12 nt is presented in Figure 1A ). The reaction conditions: [l-DNA 66 ] 0 = 5 μM; [splint] 0 = 10 μM; 20 U T4 DNA ligase in 1 × T4 ligase buffer at 20°C and 12 h. The sequences of splints were listed in Supplementary Table S1 .

    Techniques Used: Binding Assay

    12) Product Images from "Systematic analysis of the kalimantacin assembly line NRPS module using an adapted targeted mutagenesis approach"

    Article Title: Systematic analysis of the kalimantacin assembly line NRPS module using an adapted targeted mutagenesis approach

    Journal: MicrobiologyOpen

    doi: 10.1002/mbo3.326

    Ligation independent cloning strategy. Flanking regions (350 bp and 500 bp) of the 10 AA containing active site were amplified from genomic DNA with tailed primers, introducing restriction sites that enabled restriction and ligation into the pUC 18. After restriction with PstI, a mixture of linear plasmid DNA , amplified synthetic DNA fragment and T4 DNA polymerase was prepared, as proposed by Thieme et al . ( 2011 ). The mixture was incubated at 25°C for 5 min, and subsequently used for transformation of E. coli Top10 cells (Invitrogen ™ ). Correct constructs were obtained with very high efficiencies (80–95%).
    Figure Legend Snippet: Ligation independent cloning strategy. Flanking regions (350 bp and 500 bp) of the 10 AA containing active site were amplified from genomic DNA with tailed primers, introducing restriction sites that enabled restriction and ligation into the pUC 18. After restriction with PstI, a mixture of linear plasmid DNA , amplified synthetic DNA fragment and T4 DNA polymerase was prepared, as proposed by Thieme et al . ( 2011 ). The mixture was incubated at 25°C for 5 min, and subsequently used for transformation of E. coli Top10 cells (Invitrogen ™ ). Correct constructs were obtained with very high efficiencies (80–95%).

    Techniques Used: Ligation, Clone Assay, Amplification, Plasmid Preparation, Incubation, Transformation Assay, Construct

    13) Product Images from "Efficient RNA 5?-adenylation by T4 DNA ligase to facilitate practical applications"

    Article Title: Efficient RNA 5?-adenylation by T4 DNA ligase to facilitate practical applications

    Journal:

    doi: 10.1261/rna.33106

    Determining the DNA overhang requirement for RNA 5′-adenylation with T4 DNA ligase and ATP. These assays used RNA subtrate S1 (5′-GGAAGAGAUGGCGACGG-3′) and were performed in 40 mM Tris (pH 7.8), 10 mM MgCl 2 , 10 mM DTT, and 500
    Figure Legend Snippet: Determining the DNA overhang requirement for RNA 5′-adenylation with T4 DNA ligase and ATP. These assays used RNA subtrate S1 (5′-GGAAGAGAUGGCGACGG-3′) and were performed in 40 mM Tris (pH 7.8), 10 mM MgCl 2 , 10 mM DTT, and 500

    Techniques Used:

    14) Product Images from "Template-directed Chemical Ligation to Obtain 3?-3? and 5?-5? Phosphodiester DNA Linkages"

    Article Title: Template-directed Chemical Ligation to Obtain 3?-3? and 5?-5? Phosphodiester DNA Linkages

    Journal: Scientific Reports

    doi: 10.1038/srep04595

    Ligation reactions catalyzed by different mechanisms. (A) Normal 5′-3′ ligation catalyzed by DNA ligase. At the presence of template, ATP and T4 DNA ligase, two oligonucleotides were ligated together and a 5′-3′ phosphodiester bonds was formed. (B) Template-directed chemical ligation of 3′-3′ and 5′-5′ oligonucleotides activated by the coupling reagent N -Cyanoimidazole. Arrows in red represent the parallel oligonucleotide with template.
    Figure Legend Snippet: Ligation reactions catalyzed by different mechanisms. (A) Normal 5′-3′ ligation catalyzed by DNA ligase. At the presence of template, ATP and T4 DNA ligase, two oligonucleotides were ligated together and a 5′-3′ phosphodiester bonds was formed. (B) Template-directed chemical ligation of 3′-3′ and 5′-5′ oligonucleotides activated by the coupling reagent N -Cyanoimidazole. Arrows in red represent the parallel oligonucleotide with template.

    Techniques Used: Ligation

    15) 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

    16) Product Images from "Selective use of multiple vitamin D response elements underlies the 1 ?,25-dihydroxyvitamin D3-mediated negative regulation of the human CYP27B1 gene"

    Article Title: Selective use of multiple vitamin D response elements underlies the 1 ?,25-dihydroxyvitamin D3-mediated negative regulation of the human CYP27B1 gene

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm179

    DNA looping connects distal and proximal CYP27B1 promoter regions. Chromatin was extracted from HEK-293 and MCF-7 cells that had been treated for 120 min with 10 nM 1α,25(OH) 2 D 3 . The genomic DNA was digested with HinfI (recognition sites represented by vertical lines) and ligated with T4 DNA ligase. PCR was performed on purified genomic template with primer A in combination with primers B, C or D (location indicated by horizontal arrows). Digestions performed with a large subcloned CYP27B1 promoter fragment provided templates for positive control PCR reactions. Representative agarose gels of the PCR products are shown.
    Figure Legend Snippet: DNA looping connects distal and proximal CYP27B1 promoter regions. Chromatin was extracted from HEK-293 and MCF-7 cells that had been treated for 120 min with 10 nM 1α,25(OH) 2 D 3 . The genomic DNA was digested with HinfI (recognition sites represented by vertical lines) and ligated with T4 DNA ligase. PCR was performed on purified genomic template with primer A in combination with primers B, C or D (location indicated by horizontal arrows). Digestions performed with a large subcloned CYP27B1 promoter fragment provided templates for positive control PCR reactions. Representative agarose gels of the PCR products are shown.

    Techniques Used: Polymerase Chain Reaction, Purification, Positive Control

    17) Product Images from "Systematic analysis of the kalimantacin assembly line NRPS module using an adapted targeted mutagenesis approach"

    Article Title: Systematic analysis of the kalimantacin assembly line NRPS module using an adapted targeted mutagenesis approach

    Journal: MicrobiologyOpen

    doi: 10.1002/mbo3.326

    Ligation independent cloning strategy. Flanking regions (350 bp and 500 bp) of the 10 AA containing active site were amplified from genomic DNA with tailed primers, introducing restriction sites that enabled restriction and ligation into the pUC 18. After restriction with PstI, a mixture of linear plasmid DNA , amplified synthetic DNA fragment and T4 DNA polymerase was prepared, as proposed by Thieme et al . ( 2011 ). The mixture was incubated at 25°C for 5 min, and subsequently used for transformation of E. coli Top10 cells (Invitrogen ™ ). Correct constructs were obtained with very high efficiencies (80–95%).
    Figure Legend Snippet: Ligation independent cloning strategy. Flanking regions (350 bp and 500 bp) of the 10 AA containing active site were amplified from genomic DNA with tailed primers, introducing restriction sites that enabled restriction and ligation into the pUC 18. After restriction with PstI, a mixture of linear plasmid DNA , amplified synthetic DNA fragment and T4 DNA polymerase was prepared, as proposed by Thieme et al . ( 2011 ). The mixture was incubated at 25°C for 5 min, and subsequently used for transformation of E. coli Top10 cells (Invitrogen ™ ). Correct constructs were obtained with very high efficiencies (80–95%).

    Techniques Used: Ligation, Clone Assay, Amplification, Plasmid Preparation, Incubation, Transformation Assay, Construct

    18) 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

    19) 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

    20) 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

    Related Articles

    Clone Assay:

    Article Title: Truncation of the transcriptional repressor protein Cre1 in Trichoderma reesei Rut-C30 turns it into an activator
    Article Snippet: PCRs for all cloning purposes were performed with Pwo DNA Polymerase (peqlab VWR, Radnor, Pennsylvania, USA) or Phusion High-Fidelity DNA Polymerase (Thermo Scientific, Waltham, Massachusetts, USA) according to the manufacturer’s instructions. .. The PCR product was subcloned into pJET1.2 (Thermo Scientific) by blunt end ligation using T4 DNA ligase (Thermo Scientific) yielding pJET1.2-5′-cre1.

    Article Title: IRDL Cloning: A One-Tube, Zero-Background, Easy-to-Use, Directional Cloning Method Improves Throughput in Recombinant DNA Preparation
    Article Snippet: Paragraph title: One-step directional cloning of a single gene into an expression vector by the IRDL cloning ... All the reaction mixtures tested were complemented with 0.5 mM ATP and 2.5 U T4 DNA ligase (Fermentas), incubated at 37°C for 5–30 min, and immediately transformed into competent E. coli cells, with transformants subsequently selected on LB Amp+ plates.

    Amplification:

    Article Title: Truncation of the transcriptional repressor protein Cre1 in Trichoderma reesei Rut-C30 turns it into an activator
    Article Snippet: For the construction of the cre1 -96 deletion cassette the 5′-flank of cre1 -96 was amplified by PCR using chromosomal DNA of T. reesei QM6aΔtmus53 (identical sequence to Rut-C30) as template with the primers 5′cre1_NotI fwd and 5′cre1_XmaI rev. .. The PCR product was subcloned into pJET1.2 (Thermo Scientific) by blunt end ligation using T4 DNA ligase (Thermo Scientific) yielding pJET1.2-5′-cre1.

    Ligation:

    Article Title: Truncation of the transcriptional repressor protein Cre1 in Trichoderma reesei Rut-C30 turns it into an activator
    Article Snippet: For the construction of the cre1 -96 deletion cassette the 5′-flank of cre1 -96 was amplified by PCR using chromosomal DNA of T. reesei QM6aΔtmus53 (identical sequence to Rut-C30) as template with the primers 5′cre1_NotI fwd and 5′cre1_XmaI rev. .. The PCR product was subcloned into pJET1.2 (Thermo Scientific) by blunt end ligation using T4 DNA ligase (Thermo Scientific) yielding pJET1.2-5′-cre1. .. The 3′-flank of cre1 and a hygromycin B resistance cassette were amplified by PCR using chromosomal DNA of T. reesei QM6a-Cre196 [ ] as template with the primers hph_XmaI_BamHI and hph_SpeI rev and was inserted into pJET1.2 by blunt end ligation yielding pJET1.2-hph.

    Construct:

    Article Title: IRDL Cloning: A One-Tube, Zero-Background, Easy-to-Use, Directional Cloning Method Improves Throughput in Recombinant DNA Preparation
    Article Snippet: All the reaction mixtures tested were complemented with 0.5 mM ATP and 2.5 U T4 DNA ligase (Fermentas), incubated at 37°C for 5–30 min, and immediately transformed into competent E. coli cells, with transformants subsequently selected on LB Amp+ plates. .. All the reaction mixtures tested were complemented with 0.5 mM ATP and 2.5 U T4 DNA ligase (Fermentas), incubated at 37°C for 5–30 min, and immediately transformed into competent E. coli cells, with transformants subsequently selected on LB Amp+ plates.

    Polymerase Chain Reaction:

    Article Title: Truncation of the transcriptional repressor protein Cre1 in Trichoderma reesei Rut-C30 turns it into an activator
    Article Snippet: For the construction of the cre1 -96 deletion cassette the 5′-flank of cre1 -96 was amplified by PCR using chromosomal DNA of T. reesei QM6aΔtmus53 (identical sequence to Rut-C30) as template with the primers 5′cre1_NotI fwd and 5′cre1_XmaI rev. .. The PCR product was subcloned into pJET1.2 (Thermo Scientific) by blunt end ligation using T4 DNA ligase (Thermo Scientific) yielding pJET1.2-5′-cre1. .. The 3′-flank of cre1 and a hygromycin B resistance cassette were amplified by PCR using chromosomal DNA of T. reesei QM6a-Cre196 [ ] as template with the primers hph_XmaI_BamHI and hph_SpeI rev and was inserted into pJET1.2 by blunt end ligation yielding pJET1.2-hph.

    Article Title: IRDL Cloning: A One-Tube, Zero-Background, Easy-to-Use, Directional Cloning Method Improves Throughput in Recombinant DNA Preparation
    Article Snippet: Further, 48 colonies on LB plates were randomly sorted out for PCR analysis, resulting in the PCR products of all 48 colonies presented expected size bands (see ). .. All the reaction mixtures tested were complemented with 0.5 mM ATP and 2.5 U T4 DNA ligase (Fermentas), incubated at 37°C for 5–30 min, and immediately transformed into competent E. coli cells, with transformants subsequently selected on LB Amp+ plates.

    Incubation:

    Article Title: IRDL Cloning: A One-Tube, Zero-Background, Easy-to-Use, Directional Cloning Method Improves Throughput in Recombinant DNA Preparation
    Article Snippet: Apart from FastDigest restriction enzyme and digestion buffer (ingredients not disclosed by supplier), QuickCut restriction enzyme and digestion buffer (ingredients not disclosed by supplier) and restriction enzyme from New England BioLabs and CutSmart digestion buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 µg/mL BSA, pH 7.9, 25°C) were also tested ( ). .. All the reaction mixtures tested were complemented with 0.5 mM ATP and 2.5 U T4 DNA ligase (Fermentas), incubated at 37°C for 5–30 min, and immediately transformed into competent E. coli cells, with transformants subsequently selected on LB Amp+ plates. .. Restriction enzyme from different companies did display significant differences in colony number (ranging from 0.7–2×103 ).

    Expressing:

    Article Title: IRDL Cloning: A One-Tube, Zero-Background, Easy-to-Use, Directional Cloning Method Improves Throughput in Recombinant DNA Preparation
    Article Snippet: Paragraph title: One-step directional cloning of a single gene into an expression vector by the IRDL cloning ... All the reaction mixtures tested were complemented with 0.5 mM ATP and 2.5 U T4 DNA ligase (Fermentas), incubated at 37°C for 5–30 min, and immediately transformed into competent E. coli cells, with transformants subsequently selected on LB Amp+ plates.

    Sequencing:

    Article Title: Truncation of the transcriptional repressor protein Cre1 in Trichoderma reesei Rut-C30 turns it into an activator
    Article Snippet: For the construction of the cre1 -96 deletion cassette the 5′-flank of cre1 -96 was amplified by PCR using chromosomal DNA of T. reesei QM6aΔtmus53 (identical sequence to Rut-C30) as template with the primers 5′cre1_NotI fwd and 5′cre1_XmaI rev. .. The PCR product was subcloned into pJET1.2 (Thermo Scientific) by blunt end ligation using T4 DNA ligase (Thermo Scientific) yielding pJET1.2-5′-cre1.

    Article Title: IRDL Cloning: A One-Tube, Zero-Background, Easy-to-Use, Directional Cloning Method Improves Throughput in Recombinant DNA Preparation
    Article Snippet: All the reaction mixtures tested were complemented with 0.5 mM ATP and 2.5 U T4 DNA ligase (Fermentas), incubated at 37°C for 5–30 min, and immediately transformed into competent E. coli cells, with transformants subsequently selected on LB Amp+ plates. .. All the reaction mixtures tested were complemented with 0.5 mM ATP and 2.5 U T4 DNA ligase (Fermentas), incubated at 37°C for 5–30 min, and immediately transformed into competent E. coli cells, with transformants subsequently selected on LB Amp+ plates.

    Transformation Assay:

    Article Title: Truncation of the transcriptional repressor protein Cre1 in Trichoderma reesei Rut-C30 turns it into an activator
    Article Snippet: Generation of competent E. coli cells and subsequent transformation was performed according to standard protocols using CaCl2 . .. The PCR product was subcloned into pJET1.2 (Thermo Scientific) by blunt end ligation using T4 DNA ligase (Thermo Scientific) yielding pJET1.2-5′-cre1.

    Article Title: IRDL Cloning: A One-Tube, Zero-Background, Easy-to-Use, Directional Cloning Method Improves Throughput in Recombinant DNA Preparation
    Article Snippet: Apart from FastDigest restriction enzyme and digestion buffer (ingredients not disclosed by supplier), QuickCut restriction enzyme and digestion buffer (ingredients not disclosed by supplier) and restriction enzyme from New England BioLabs and CutSmart digestion buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 µg/mL BSA, pH 7.9, 25°C) were also tested ( ). .. All the reaction mixtures tested were complemented with 0.5 mM ATP and 2.5 U T4 DNA ligase (Fermentas), incubated at 37°C for 5–30 min, and immediately transformed into competent E. coli cells, with transformants subsequently selected on LB Amp+ plates. .. Restriction enzyme from different companies did display significant differences in colony number (ranging from 0.7–2×103 ).

    Plasmid Preparation:

    Article Title: Truncation of the transcriptional repressor protein Cre1 in Trichoderma reesei Rut-C30 turns it into an activator
    Article Snippet: Paragraph title: Plasmid construction ... The PCR product was subcloned into pJET1.2 (Thermo Scientific) by blunt end ligation using T4 DNA ligase (Thermo Scientific) yielding pJET1.2-5′-cre1.

    Article Title: IRDL Cloning: A One-Tube, Zero-Background, Easy-to-Use, Directional Cloning Method Improves Throughput in Recombinant DNA Preparation
    Article Snippet: Paragraph title: One-step directional cloning of a single gene into an expression vector by the IRDL cloning ... All the reaction mixtures tested were complemented with 0.5 mM ATP and 2.5 U T4 DNA ligase (Fermentas), incubated at 37°C for 5–30 min, and immediately transformed into competent E. coli cells, with transformants subsequently selected on LB Amp+ plates.

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    • t4 dna ligase  (Thermo Fisher)
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    Thermo Fisher t4 dna ligase
    Effects of the distance between a hairpin and the ligation site on the magnitude of ‘terminal hairpin effect’ for the selective formation of single-stranded DNA ring. ( A ) The solution structures of l-DNAs used here. ( B ) Lane 1, L64 2-4,23-4,51-2 without the treatment; lane 2, L64 2-4,23-4,51-2 treated with <t>T4</t> DNA ligase in the presence of 12-nt splint which is complementary with the 6-nt sequences in the 3′- and 5′-ends of l-DNA; lane 3, L64 3-4,24-4 alone; lane 4, L64 3-4,24-4 treated with T4 DNA ligase in the presence of 12-nt splint; lane 5, L64 4-4,25-4 alone; lane 6, L64 4-4,25-4 treated with T4 DNA ligase in the presence of 12-nt splint; lane 7, L64 5-4,26-4 alone; lane 8, L64 5–4,26-4 treated with T4 DNA ligase in the presence of 12-nt splint. lane 9, L64 6-4,27-4 alone; lane 10, L64 6-4,27-4 treated with T4 DNA ligase in the presence of 12-nt splint. Reaction conditions are the same as described in Figure 2 .
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 75/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ubr5  (Thermo Fisher)
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    Thermo Fisher ubr5
    Pull-down assays of HECT truncations with PEPCK1. (A) GST-PEPCK pull-down with three <t>UBR5</t> truncation derivatives. Result shows that the HECT domain is sufficient to interact with PEPCK1. (B) Pull-down assays of HECT N-lobe (Flag-UBR5-9) and HECT C-lobe (Flag-UBR5-10) with GST-PEPCK1. Result shows that the N-lobe has a strong interaction with PEPCK1 while C-lobe does not.
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    Effects of the distance between a hairpin and the ligation site on the magnitude of ‘terminal hairpin effect’ for the selective formation of single-stranded DNA ring. ( A ) The solution structures of l-DNAs used here. ( B ) Lane 1, L64 2-4,23-4,51-2 without the treatment; lane 2, L64 2-4,23-4,51-2 treated with T4 DNA ligase in the presence of 12-nt splint which is complementary with the 6-nt sequences in the 3′- and 5′-ends of l-DNA; lane 3, L64 3-4,24-4 alone; lane 4, L64 3-4,24-4 treated with T4 DNA ligase in the presence of 12-nt splint; lane 5, L64 4-4,25-4 alone; lane 6, L64 4-4,25-4 treated with T4 DNA ligase in the presence of 12-nt splint; lane 7, L64 5-4,26-4 alone; lane 8, L64 5–4,26-4 treated with T4 DNA ligase in the presence of 12-nt splint. lane 9, L64 6-4,27-4 alone; lane 10, L64 6-4,27-4 treated with T4 DNA ligase in the presence of 12-nt splint. Reaction conditions are the same as described in Figure 2 .

    Journal: Nucleic Acids Research

    Article Title: Terminal hairpin in oligonucleotide dominantly prioritizes intramolecular cyclization by T4 ligase over intermolecular polymerization: an exclusive methodology for producing ssDNA rings

    doi: 10.1093/nar/gky769

    Figure Lengend Snippet: Effects of the distance between a hairpin and the ligation site on the magnitude of ‘terminal hairpin effect’ for the selective formation of single-stranded DNA ring. ( A ) The solution structures of l-DNAs used here. ( B ) Lane 1, L64 2-4,23-4,51-2 without the treatment; lane 2, L64 2-4,23-4,51-2 treated with T4 DNA ligase in the presence of 12-nt splint which is complementary with the 6-nt sequences in the 3′- and 5′-ends of l-DNA; lane 3, L64 3-4,24-4 alone; lane 4, L64 3-4,24-4 treated with T4 DNA ligase in the presence of 12-nt splint; lane 5, L64 4-4,25-4 alone; lane 6, L64 4-4,25-4 treated with T4 DNA ligase in the presence of 12-nt splint; lane 7, L64 5-4,26-4 alone; lane 8, L64 5–4,26-4 treated with T4 DNA ligase in the presence of 12-nt splint. lane 9, L64 6-4,27-4 alone; lane 10, L64 6-4,27-4 treated with T4 DNA ligase in the presence of 12-nt splint. Reaction conditions are the same as described in Figure 2 .

    Article Snippet: T4 DNA ligase, Exonuclease I and SYBR Green II were also obtained from Thermo Scientific (Pittsburgh, PA, USA).

    Techniques: Ligation

    Effects of the stability of hairpin on the cyclization by T4 DNA ligase. ( A ) The solution conformations of L64 1-4,24-4 , L64 1-6,24-4 , L64 1-7,24-4 and L60 1-7,20-4 , determined by Mfold calculation. ( B ) Lane 1, L64 1-4,24-4 without T4 ligase treatment; lane 2, L64 1-4,24-4 with T4 ligase treatment; lane 3, L64 1-6,24-4 alone; lane 4, L64 1-6,24-4 with T4 ligase treatment; lane 5, L64 1-7,24-4 alone; lane 6, L64 1-7,24-4 with T4 ligase treatment; lane 7, L60 1-7,20-4 alone; lane 8, L60 1-7,20-4 with T4 ligase treatment. The enzymatic conditions are the same as described in Figure 2 .

    Journal: Nucleic Acids Research

    Article Title: Terminal hairpin in oligonucleotide dominantly prioritizes intramolecular cyclization by T4 ligase over intermolecular polymerization: an exclusive methodology for producing ssDNA rings

    doi: 10.1093/nar/gky769

    Figure Lengend Snippet: Effects of the stability of hairpin on the cyclization by T4 DNA ligase. ( A ) The solution conformations of L64 1-4,24-4 , L64 1-6,24-4 , L64 1-7,24-4 and L60 1-7,20-4 , determined by Mfold calculation. ( B ) Lane 1, L64 1-4,24-4 without T4 ligase treatment; lane 2, L64 1-4,24-4 with T4 ligase treatment; lane 3, L64 1-6,24-4 alone; lane 4, L64 1-6,24-4 with T4 ligase treatment; lane 5, L64 1-7,24-4 alone; lane 6, L64 1-7,24-4 with T4 ligase treatment; lane 7, L60 1-7,20-4 alone; lane 8, L60 1-7,20-4 with T4 ligase treatment. The enzymatic conditions are the same as described in Figure 2 .

    Article Snippet: T4 DNA ligase, Exonuclease I and SYBR Green II were also obtained from Thermo Scientific (Pittsburgh, PA, USA).

    Techniques:

    Terminal hairpin strategy for T4 DNA ligase-mediated preparation of DNA rings of smaller sizes. ( A ) Solution structures of 74-, 64-, 54-, 44- and 34-nt l-DNAs. ( B ) Gel electrophoresis patterns of the T4 ligase ligation products. The conditions of T4 ligase reactions are the same as described in Figure 2 .

    Journal: Nucleic Acids Research

    Article Title: Terminal hairpin in oligonucleotide dominantly prioritizes intramolecular cyclization by T4 ligase over intermolecular polymerization: an exclusive methodology for producing ssDNA rings

    doi: 10.1093/nar/gky769

    Figure Lengend Snippet: Terminal hairpin strategy for T4 DNA ligase-mediated preparation of DNA rings of smaller sizes. ( A ) Solution structures of 74-, 64-, 54-, 44- and 34-nt l-DNAs. ( B ) Gel electrophoresis patterns of the T4 ligase ligation products. The conditions of T4 ligase reactions are the same as described in Figure 2 .

    Article Snippet: T4 DNA ligase, Exonuclease I and SYBR Green II were also obtained from Thermo Scientific (Pittsburgh, PA, USA).

    Techniques: Nucleic Acid Electrophoresis, Ligation

    Dominant cyclization of l-DNA using hairpins as internal promoters. ( A ) The solution structures of L64 3-4,24-4 , L64 16-4,37-4 and L64 3-4 , determined by Mfold calculation under the conditions of [Mg 2+ ] = 10 mM and 25°C. ( B ) Treatments of these l-DNAs with T4 DNA ligase. Lane 1, L64 3-4,24-4 without the T4 ligase treatment; lane 2, L64 3-4,24-4 treated with T4 DNA ligase in the presence of 12-nt splint which is complementary with the 6-nt sequences in the 3′- and 5′-ends of L64 3-4,24–4 ; lane 4, L64 16-4,37-4 without the treatment; lane 5, L64 16-4,37-4 treated with T4 DNA ligase in the presence of 12-nt splint. Lane 7, L64 3-4 without the treatment; lane 8, L64 3-4 treated with T4 DNA ligase in the presence of 12-nt splint. In lanes 3, 6 and 9, the products in lanes 2, 5 and 8 were further treated with Exonuclease I to remove non-cyclic products. The conditions for the T4 ligase reactions: [l-DNA] 0 = 5 μM, [splint] 0 = 10 μM and 10 U T4 DNA ligase in 1× T4 DNA ligase buffer at 25°C for 12 h.

    Journal: Nucleic Acids Research

    Article Title: Terminal hairpin in oligonucleotide dominantly prioritizes intramolecular cyclization by T4 ligase over intermolecular polymerization: an exclusive methodology for producing ssDNA rings

    doi: 10.1093/nar/gky769

    Figure Lengend Snippet: Dominant cyclization of l-DNA using hairpins as internal promoters. ( A ) The solution structures of L64 3-4,24-4 , L64 16-4,37-4 and L64 3-4 , determined by Mfold calculation under the conditions of [Mg 2+ ] = 10 mM and 25°C. ( B ) Treatments of these l-DNAs with T4 DNA ligase. Lane 1, L64 3-4,24-4 without the T4 ligase treatment; lane 2, L64 3-4,24-4 treated with T4 DNA ligase in the presence of 12-nt splint which is complementary with the 6-nt sequences in the 3′- and 5′-ends of L64 3-4,24–4 ; lane 4, L64 16-4,37-4 without the treatment; lane 5, L64 16-4,37-4 treated with T4 DNA ligase in the presence of 12-nt splint. Lane 7, L64 3-4 without the treatment; lane 8, L64 3-4 treated with T4 DNA ligase in the presence of 12-nt splint. In lanes 3, 6 and 9, the products in lanes 2, 5 and 8 were further treated with Exonuclease I to remove non-cyclic products. The conditions for the T4 ligase reactions: [l-DNA] 0 = 5 μM, [splint] 0 = 10 μM and 10 U T4 DNA ligase in 1× T4 DNA ligase buffer at 25°C for 12 h.

    Article Snippet: T4 DNA ligase, Exonuclease I and SYBR Green II were also obtained from Thermo Scientific (Pittsburgh, PA, USA).

    Techniques:

    Highly selective cyclization at unusually high substrate concentrations using terminal hairpin strategy. Lane 1, L64 3-4,24-4 without T4 treatment; lane 2, T4 reaction at [L64 3-4,24-4 ] 0 = 10 μM; lane 3, [L64 3-4,24-4 ] 0 = 20 μM; lane 4, [L64 3-4,24-4 ] 0 = 40 μM; lane 5, [L64 3-4,24-4 ] 0 = 60 μM; lane 6, [L64 3-4,24-4 ] 0 = 100 μM. In lanes 8 - 10, L64 16-4,37-4 having no terminal hairpin is used. Lane 8, L64 16-4,37-4 without T4 treatment; lane 9, [L64 16-4,37-4 ] 0 = 100 μM. Reaction conditions: [l-DNA] 0 /[splint] 0 = 1/2 and 10 U T4 DNA ligase in 1 × T4 DNA ligase buffer at 25°C. In lanes 7 and 10, 0.1× T4 DNA ligase buffer was used in place of 1× T4 buffer, according to ref. ( 30 ) (see text for details).

    Journal: Nucleic Acids Research

    Article Title: Terminal hairpin in oligonucleotide dominantly prioritizes intramolecular cyclization by T4 ligase over intermolecular polymerization: an exclusive methodology for producing ssDNA rings

    doi: 10.1093/nar/gky769

    Figure Lengend Snippet: Highly selective cyclization at unusually high substrate concentrations using terminal hairpin strategy. Lane 1, L64 3-4,24-4 without T4 treatment; lane 2, T4 reaction at [L64 3-4,24-4 ] 0 = 10 μM; lane 3, [L64 3-4,24-4 ] 0 = 20 μM; lane 4, [L64 3-4,24-4 ] 0 = 40 μM; lane 5, [L64 3-4,24-4 ] 0 = 60 μM; lane 6, [L64 3-4,24-4 ] 0 = 100 μM. In lanes 8 - 10, L64 16-4,37-4 having no terminal hairpin is used. Lane 8, L64 16-4,37-4 without T4 treatment; lane 9, [L64 16-4,37-4 ] 0 = 100 μM. Reaction conditions: [l-DNA] 0 /[splint] 0 = 1/2 and 10 U T4 DNA ligase in 1 × T4 DNA ligase buffer at 25°C. In lanes 7 and 10, 0.1× T4 DNA ligase buffer was used in place of 1× T4 buffer, according to ref. ( 30 ) (see text for details).

    Article Snippet: T4 DNA ligase, Exonuclease I and SYBR Green II were also obtained from Thermo Scientific (Pittsburgh, PA, USA).

    Techniques:

    Comparison of the reaction conversion and yield of monomeric cyclic ring between substrates with and without the terminal hairpin. ( A ) Time-courses for the T4 ligase-mediated ligation of L64 3-4,24-4 (circles) and L64 16-4,37-4 (rectangles). The total amounts of DNA, consumed in the presence of T4 ligase (by both intramolecular and intermolecular ligation), are plotted as a function of reaction time. In ( B ), the yield of DNA ring is shown as a function of reaction time. Reaction conditions: [l-DNA] 0 = 5 μM, [splint] 0 = 10 μM, and 10 U T4 DNA ligase in 1× T4 DNA ligase buffer at 25°C.

    Journal: Nucleic Acids Research

    Article Title: Terminal hairpin in oligonucleotide dominantly prioritizes intramolecular cyclization by T4 ligase over intermolecular polymerization: an exclusive methodology for producing ssDNA rings

    doi: 10.1093/nar/gky769

    Figure Lengend Snippet: Comparison of the reaction conversion and yield of monomeric cyclic ring between substrates with and without the terminal hairpin. ( A ) Time-courses for the T4 ligase-mediated ligation of L64 3-4,24-4 (circles) and L64 16-4,37-4 (rectangles). The total amounts of DNA, consumed in the presence of T4 ligase (by both intramolecular and intermolecular ligation), are plotted as a function of reaction time. In ( B ), the yield of DNA ring is shown as a function of reaction time. Reaction conditions: [l-DNA] 0 = 5 μM, [splint] 0 = 10 μM, and 10 U T4 DNA ligase in 1× T4 DNA ligase buffer at 25°C.

    Article Snippet: T4 DNA ligase, Exonuclease I and SYBR Green II were also obtained from Thermo Scientific (Pittsburgh, PA, USA).

    Techniques: Ligation

    Cyclization of l-DNA 66 by T4 DNA ligase. Schematic views of the formation of ( A ) single-stranded DNA ring (c-DNA 66 ) with the assistance of splint-12 nt and ( B ) polymers from multiple l-DNA 66 strands. The single-stranded DNA substrate (l-DNA 66 ) bears a phosphate at the 5′-terminus. ( C ) Effects of [l-DNA 66 ] 0 on the formation of c-DNA 66 and the polymers in 1× T4 ligase buffer (conventional method). The selectivity for the formation of c-DNA 66 is presented below the corresponding band. [l-DNA 66 ] 0 /[splint-12 nt] 0 = 1/2 at 20°C for 12 h. All the DNA substrate was added to the mixture all at once at the beginning of the reaction.

    Journal: Nucleic Acids Research

    Article Title: Highly efficient preparation of single-stranded DNA rings by T4 ligase at abnormally low Mg(II) concentration

    doi: 10.1093/nar/gkx553

    Figure Lengend Snippet: Cyclization of l-DNA 66 by T4 DNA ligase. Schematic views of the formation of ( A ) single-stranded DNA ring (c-DNA 66 ) with the assistance of splint-12 nt and ( B ) polymers from multiple l-DNA 66 strands. The single-stranded DNA substrate (l-DNA 66 ) bears a phosphate at the 5′-terminus. ( C ) Effects of [l-DNA 66 ] 0 on the formation of c-DNA 66 and the polymers in 1× T4 ligase buffer (conventional method). The selectivity for the formation of c-DNA 66 is presented below the corresponding band. [l-DNA 66 ] 0 /[splint-12 nt] 0 = 1/2 at 20°C for 12 h. All the DNA substrate was added to the mixture all at once at the beginning of the reaction.

    Article Snippet: T4 DNA ligase was purchased from Thermo Scientific (Pittsburgh, PA, USA), together with the 10 × T4 ligase buffer.

    Techniques:

    Effects of ( A ) [Mg 2+ ] 0 and ( B ) [ATP] 0 on the cyclization of l-DNA 66 by T4 DNA ligase. [l-DNA 66 ] 0 = 1 μM, [splint-12 nt] 0 = 2 μM, 5 U T4 DNA ligase at 20°C and 12 h. [ATP] 0 = 25 μM, [DTT] = 0.5 mM, and [Tris–HCl] = 2 mM in ( A ), whereas [MgCl 2 ] = 0.5 mM, [DTT] = 0.5 mM, and [Tris–HCl] = 2 mM in ( B ).

    Journal: Nucleic Acids Research

    Article Title: Highly efficient preparation of single-stranded DNA rings by T4 ligase at abnormally low Mg(II) concentration

    doi: 10.1093/nar/gkx553

    Figure Lengend Snippet: Effects of ( A ) [Mg 2+ ] 0 and ( B ) [ATP] 0 on the cyclization of l-DNA 66 by T4 DNA ligase. [l-DNA 66 ] 0 = 1 μM, [splint-12 nt] 0 = 2 μM, 5 U T4 DNA ligase at 20°C and 12 h. [ATP] 0 = 25 μM, [DTT] = 0.5 mM, and [Tris–HCl] = 2 mM in ( A ), whereas [MgCl 2 ] = 0.5 mM, [DTT] = 0.5 mM, and [Tris–HCl] = 2 mM in ( B ).

    Article Snippet: T4 DNA ligase was purchased from Thermo Scientific (Pittsburgh, PA, USA), together with the 10 × T4 ligase buffer.

    Techniques:

    Effects of the concentration of T4 ligase buffer on the efficiencies of cyclization of l-DNA 66 and its polymerization. All the DNA substrate was added to the mixture all at once at the beginning of the reaction. The selectivity for the formation of c-DNA 66 is presented below the corresponding band. The reaction conditions: [l-DNA 66 ] 0 = 1 μM; [splint-12 nt] 0 = 2 μM; 5 U T4 DNA ligase at 20°C and 12 h. Note that 1 × T4 ligase buffer contains 10 mM MgCl 2 , 500 μM ATP, 10 mM DTT and 40 mM Tris-HCl.

    Journal: Nucleic Acids Research

    Article Title: Highly efficient preparation of single-stranded DNA rings by T4 ligase at abnormally low Mg(II) concentration

    doi: 10.1093/nar/gkx553

    Figure Lengend Snippet: Effects of the concentration of T4 ligase buffer on the efficiencies of cyclization of l-DNA 66 and its polymerization. All the DNA substrate was added to the mixture all at once at the beginning of the reaction. The selectivity for the formation of c-DNA 66 is presented below the corresponding band. The reaction conditions: [l-DNA 66 ] 0 = 1 μM; [splint-12 nt] 0 = 2 μM; 5 U T4 DNA ligase at 20°C and 12 h. Note that 1 × T4 ligase buffer contains 10 mM MgCl 2 , 500 μM ATP, 10 mM DTT and 40 mM Tris-HCl.

    Article Snippet: T4 DNA ligase was purchased from Thermo Scientific (Pittsburgh, PA, USA), together with the 10 × T4 ligase buffer.

    Techniques: Concentration Assay

    Effects of the length of splints on the efficiencies of cyclization of l-DNA 66 and its polymerization under conventional conditions. All the DNA substrate was added to the mixture all at once at the beginning of the reaction. Each of the splints is complementary to equal number of nucleotides in the 5′- and 3′-ends of l-DNA 66 , respectively (the binding mode of splint-12 nt is presented in Figure 1A ). The reaction conditions: [l-DNA 66 ] 0 = 5 μM; [splint] 0 = 10 μM; 20 U T4 DNA ligase in 1 × T4 ligase buffer at 20°C and 12 h. The sequences of splints were listed in Supplementary Table S1 .

    Journal: Nucleic Acids Research

    Article Title: Highly efficient preparation of single-stranded DNA rings by T4 ligase at abnormally low Mg(II) concentration

    doi: 10.1093/nar/gkx553

    Figure Lengend Snippet: Effects of the length of splints on the efficiencies of cyclization of l-DNA 66 and its polymerization under conventional conditions. All the DNA substrate was added to the mixture all at once at the beginning of the reaction. Each of the splints is complementary to equal number of nucleotides in the 5′- and 3′-ends of l-DNA 66 , respectively (the binding mode of splint-12 nt is presented in Figure 1A ). The reaction conditions: [l-DNA 66 ] 0 = 5 μM; [splint] 0 = 10 μM; 20 U T4 DNA ligase in 1 × T4 ligase buffer at 20°C and 12 h. The sequences of splints were listed in Supplementary Table S1 .

    Article Snippet: T4 DNA ligase was purchased from Thermo Scientific (Pittsburgh, PA, USA), together with the 10 × T4 ligase buffer.

    Techniques: Binding Assay

    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: Ligation was performed in 80 μl reactions containing 1 × T4 RNA ligase buffer, 20% PEG-8000, 0.5 mM ATP, 10/20 pmol of adapter splinter mix CL78/TL38, 1, 2 or 4 pmol acceptor oligonucleotide and 30 U T4 DNA ligase (ThermoFisher Scientific).

    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: Ligation was performed in 80 μl reactions containing 1 × T4 RNA ligase buffer, 20% PEG-8000, 0.5 mM ATP, 10/20 pmol of adapter splinter mix CL78/TL38, 1, 2 or 4 pmol acceptor oligonucleotide and 30 U T4 DNA ligase (ThermoFisher Scientific).

    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: Ligation was performed in 80 μl reactions containing 1 × T4 RNA ligase buffer, 20% PEG-8000, 0.5 mM ATP, 10/20 pmol of adapter splinter mix CL78/TL38, 1, 2 or 4 pmol acceptor oligonucleotide and 30 U T4 DNA ligase (ThermoFisher Scientific).

    Techniques: Ligation, Sequencing, Ancient DNA Assay, Gas Chromatography

    Pull-down assays of HECT truncations with PEPCK1. (A) GST-PEPCK pull-down with three UBR5 truncation derivatives. Result shows that the HECT domain is sufficient to interact with PEPCK1. (B) Pull-down assays of HECT N-lobe (Flag-UBR5-9) and HECT C-lobe (Flag-UBR5-10) with GST-PEPCK1. Result shows that the N-lobe has a strong interaction with PEPCK1 while C-lobe does not.

    Journal: Biology Open

    Article Title: Characterization of interaction and ubiquitination of phosphoenolpyruvate carboxykinase by E3 ligase UBR5

    doi: 10.1242/bio.037366

    Figure Lengend Snippet: Pull-down assays of HECT truncations with PEPCK1. (A) GST-PEPCK pull-down with three UBR5 truncation derivatives. Result shows that the HECT domain is sufficient to interact with PEPCK1. (B) Pull-down assays of HECT N-lobe (Flag-UBR5-9) and HECT C-lobe (Flag-UBR5-10) with GST-PEPCK1. Result shows that the N-lobe has a strong interaction with PEPCK1 while C-lobe does not.

    Article Snippet: The cDNA of truncations was amplified from human UBR5 gene (GenBank ID: NM_015902 ) or human PEPCK1 gene (GenBank ID: BC023978 ) by PCR with desired restriction sites.

    Techniques:

    Lys243 and Lys342 in PEPCK1 are the ubiquitination sites. (A) Ubiquitination assays of K243A and K342A. K243 and K342 seem to be the two ubiquitination sites of PEPCK1. (B) Pull-downs assays of K243A and K342A mutants. Pull-down assays indicate that these two ubiquitination sites do not affect the interaction between PEPCK1 and UBR5.

    Journal: Biology Open

    Article Title: Characterization of interaction and ubiquitination of phosphoenolpyruvate carboxykinase by E3 ligase UBR5

    doi: 10.1242/bio.037366

    Figure Lengend Snippet: Lys243 and Lys342 in PEPCK1 are the ubiquitination sites. (A) Ubiquitination assays of K243A and K342A. K243 and K342 seem to be the two ubiquitination sites of PEPCK1. (B) Pull-downs assays of K243A and K342A mutants. Pull-down assays indicate that these two ubiquitination sites do not affect the interaction between PEPCK1 and UBR5.

    Article Snippet: The cDNA of truncations was amplified from human UBR5 gene (GenBank ID: NM_015902 ) or human PEPCK1 gene (GenBank ID: BC023978 ) by PCR with desired restriction sites.

    Techniques:

    Pull-down assays of PEPCK1 and its mutants. (A) GST-HECT pull-down with PEPCK1 and its mutants (3K/Q, 3K/R, K70R, K71R, K594R) in vitro . (B) GST-HECT pull-down with PEPCK1 and its mutants (3K/Q, 3K/R, K70R, K71R, K594R) in vivo . Results indicate that the mutants of these three acetylation sites of PEPCK1 do not affect its binding with UBR5.

    Journal: Biology Open

    Article Title: Characterization of interaction and ubiquitination of phosphoenolpyruvate carboxykinase by E3 ligase UBR5

    doi: 10.1242/bio.037366

    Figure Lengend Snippet: Pull-down assays of PEPCK1 and its mutants. (A) GST-HECT pull-down with PEPCK1 and its mutants (3K/Q, 3K/R, K70R, K71R, K594R) in vitro . (B) GST-HECT pull-down with PEPCK1 and its mutants (3K/Q, 3K/R, K70R, K71R, K594R) in vivo . Results indicate that the mutants of these three acetylation sites of PEPCK1 do not affect its binding with UBR5.

    Article Snippet: The cDNA of truncations was amplified from human UBR5 gene (GenBank ID: NM_015902 ) or human PEPCK1 gene (GenBank ID: BC023978 ) by PCR with desired restriction sites.

    Techniques: In Vitro, In Vivo, Binding Assay

    The role of two ubiquitination sites (K243 and K342) in the interaction between PEPCK1 and UBR5. (A) Schematic diagram of PEPCK1 and UBR5. UBR5 interacts with PEPCK1 through the HECT N-lobe, and catalyzes the PEPCK1 ubiquitination of K243 or K342 through C2468. The interaction site and catalytic site are of a two-versus-two relationship. (B) Location of the two ubiquitination sites of PEPCK1. The structure of PEPCK1 is shown as surface representation (gray). The two ubiquitination sites (K243 and K342) are shown as sticks and colored by elements. The two lysine residues are positioned at two opposite sides flanking the PEPCK1 active site and exposed to the surface, which makes them amendable to be ubiquitinated by UBR5.

    Journal: Biology Open

    Article Title: Characterization of interaction and ubiquitination of phosphoenolpyruvate carboxykinase by E3 ligase UBR5

    doi: 10.1242/bio.037366

    Figure Lengend Snippet: The role of two ubiquitination sites (K243 and K342) in the interaction between PEPCK1 and UBR5. (A) Schematic diagram of PEPCK1 and UBR5. UBR5 interacts with PEPCK1 through the HECT N-lobe, and catalyzes the PEPCK1 ubiquitination of K243 or K342 through C2468. The interaction site and catalytic site are of a two-versus-two relationship. (B) Location of the two ubiquitination sites of PEPCK1. The structure of PEPCK1 is shown as surface representation (gray). The two ubiquitination sites (K243 and K342) are shown as sticks and colored by elements. The two lysine residues are positioned at two opposite sides flanking the PEPCK1 active site and exposed to the surface, which makes them amendable to be ubiquitinated by UBR5.

    Article Snippet: The cDNA of truncations was amplified from human UBR5 gene (GenBank ID: NM_015902 ) or human PEPCK1 gene (GenBank ID: BC023978 ) by PCR with desired restriction sites.

    Techniques:

    Pull-down and ubiquitination assays of PEPCK ΔN and ΔC in vivo . (A) GST-HECT pull-down with PEPCK ΔN (74-622 aa) and ΔC (1-560 aa). (B) Ubiquitination assays of these two PEPCK truncations expressed in HEK293T cells. Results indicate that PEPCK1 truncations which feature the loss of acetylation sites still could be recognized and ubiquitinated by UBR5.

    Journal: Biology Open

    Article Title: Characterization of interaction and ubiquitination of phosphoenolpyruvate carboxykinase by E3 ligase UBR5

    doi: 10.1242/bio.037366

    Figure Lengend Snippet: Pull-down and ubiquitination assays of PEPCK ΔN and ΔC in vivo . (A) GST-HECT pull-down with PEPCK ΔN (74-622 aa) and ΔC (1-560 aa). (B) Ubiquitination assays of these two PEPCK truncations expressed in HEK293T cells. Results indicate that PEPCK1 truncations which feature the loss of acetylation sites still could be recognized and ubiquitinated by UBR5.

    Article Snippet: The cDNA of truncations was amplified from human UBR5 gene (GenBank ID: NM_015902 ) or human PEPCK1 gene (GenBank ID: BC023978 ) by PCR with desired restriction sites.

    Techniques: In Vivo

    Purification of UBR5-2-His and UBR5-5-His. These two proteins were purified by HiLoad 16/600 Superdex 200 pg, in which the column volume was ∼120 ml. Purified protein samples were analyzed by SDS-PAGE. (A) SDS-PAGE shows that UBR5-2-His truncation is of high purity with a molecular weight of ∼31 kDa. The retention volume of UBR5-1 truncation is 63.3 ml, which indicates that UBR5-2 truncation is oligomer. (B) UBR5-5-His is of good purity with little degradation. This truncation acts as a 64 kDa monomer, corresponding to the 79.8 ml retention volume.

    Journal: Biology Open

    Article Title: Characterization of interaction and ubiquitination of phosphoenolpyruvate carboxykinase by E3 ligase UBR5

    doi: 10.1242/bio.037366

    Figure Lengend Snippet: Purification of UBR5-2-His and UBR5-5-His. These two proteins were purified by HiLoad 16/600 Superdex 200 pg, in which the column volume was ∼120 ml. Purified protein samples were analyzed by SDS-PAGE. (A) SDS-PAGE shows that UBR5-2-His truncation is of high purity with a molecular weight of ∼31 kDa. The retention volume of UBR5-1 truncation is 63.3 ml, which indicates that UBR5-2 truncation is oligomer. (B) UBR5-5-His is of good purity with little degradation. This truncation acts as a 64 kDa monomer, corresponding to the 79.8 ml retention volume.

    Article Snippet: The cDNA of truncations was amplified from human UBR5 gene (GenBank ID: NM_015902 ) or human PEPCK1 gene (GenBank ID: BC023978 ) by PCR with desired restriction sites.

    Techniques: Purification, SDS Page, Molecular Weight