t4 dna ligase  (Thermo Fisher)


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    Name:
    T4 DNA Ligase
    Description:
    Thermo Scientific T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex DNA or RNA The enzyme repairs single strand nicks in duplex DNA RNA or DNA RNA hybrids It also joins DNA fragments with either cohesive or blunt termini but has no activity on single stranded nucleic acids T4 DNA Ligase requires ATP as a cofactor Highlights• Active in Themo Scientific restriction enzyme PCR and RT buffers when supplemented with ATP • Fast sticky end ligation is completed in 10 minutes at room temperature• Supplied with PEG solution for efficient blunt end ligationApplications• Cloning of restriction enzyme generated DNA fragments• Cloning of PCR products• Joining of double stranded oligonucleotide linkers or adaptors to DNA• Site directed mutagenesis• Amplified fragment length polymorphism AFLP • Ligase mediated RNA detection see Reference 3 • Nick repair in duplex DNA RNA or DNA RNA hybrids• Self circularization of linear DNA Includes• T4 DNA Ligase• 10X T4 DNA Ligase Buffer• 50 PEG SolutionNotes• Binding of T4 DNA Ligase to DNA may result in a band shift in agarose gels To avoid this incubate samples with 6X DNA Loading Dye SDS Solution at 70°C for 5 min or 65°C for 10 minutes and chill on ice prior to electrophoresis • The volume of the ligation reaction mixture should not exceed 10 of the competent cell volume in the transformation process • Prior to electro transformation remove T4 DNA Ligase from the ligation mixture using spin columns or chloroform extraction The extracted DNA can be further precipitated with ethanol
    Catalog Number:
    EL0013
    Price:
    None
    Category:
    Proteins Enzymes Peptides
    Applications:
    Cloning|Restriction Enzyme Cloning
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    Structured Review

    Thermo Fisher t4 dna ligase
    DNA barcoding experimental scheme. Target DNA strands are immobilized on a microscope slide, and dye-labeled barcodes are introduced together with <t>T4</t> DNA ligase in the microfluidic chamber (1). Complementary barcodes bind transiently to the target site (2), whereas mismatched barcodes bind on an even shorter timescale (2′). Successful ligation is observed for the complementary barcodes (3) but not for the mismatched barcodes (3′). Ligation product shows stable binding to the target DNA (4), whereas mismatched barcodes dissociate and are washed away before imaging. To see this figure in color, go online.
    Thermo Scientific T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex DNA or RNA The enzyme repairs single strand nicks in duplex DNA RNA or DNA RNA hybrids It also joins DNA fragments with either cohesive or blunt termini but has no activity on single stranded nucleic acids T4 DNA Ligase requires ATP as a cofactor Highlights• Active in Themo Scientific restriction enzyme PCR and RT buffers when supplemented with ATP • Fast sticky end ligation is completed in 10 minutes at room temperature• Supplied with PEG solution for efficient blunt end ligationApplications• Cloning of restriction enzyme generated DNA fragments• Cloning of PCR products• Joining of double stranded oligonucleotide linkers or adaptors to DNA• Site directed mutagenesis• Amplified fragment length polymorphism AFLP • Ligase mediated RNA detection see Reference 3 • Nick repair in duplex DNA RNA or DNA RNA hybrids• Self circularization of linear DNA Includes• T4 DNA Ligase• 10X T4 DNA Ligase Buffer• 50 PEG SolutionNotes• Binding of T4 DNA Ligase to DNA may result in a band shift in agarose gels To avoid this incubate samples with 6X DNA Loading Dye SDS Solution at 70°C for 5 min or 65°C for 10 minutes and chill on ice prior to electrophoresis • The volume of the ligation reaction mixture should not exceed 10 of the competent cell volume in the transformation process • Prior to electro transformation remove T4 DNA Ligase from the ligation mixture using spin columns or chloroform extraction The extracted DNA can be further precipitated with ethanol
    https://www.bioz.com/result/t4 dna ligase/product/Thermo Fisher
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    Images

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

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

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2018.08.013

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

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

    2) 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

    3) 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

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

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

    Journal: Infection and Immunity

    doi:

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

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

    5) Product Images from "Cognate base‐pair selectivity of hydrophobic unnatural bases in DNA ligation by T4 DNA ligase. Cognate base‐pair selectivity of hydrophobic unnatural bases in DNA ligation by T4 DNA ligase"

    Article Title: Cognate base‐pair selectivity of hydrophobic unnatural bases in DNA ligation by T4 DNA ligase. Cognate base‐pair selectivity of hydrophobic unnatural bases in DNA ligation by T4 DNA ligase

    Journal: Biopolymers

    doi: 10.1002/bip.23407

    Summary of ligation efficiencies with the hydrophobic unnatural Ds base at the 5′‐end of the donor strand or at the 3′‐end of the acceptor strands. The rates of the ligation (turnover numbers) were calculated from the yields of the ligated products at 5 minutes (dsDNA: 0.5 μM; T4 DNA ligase: 0.0025 Weiss U/μL, 3.6 ± 0.9 nM) by averaging independent repetitive data (n = 2‐4, Figures 3 , S8 and S10 ), and indicated with the standard deviations
    Figure Legend Snippet: Summary of ligation efficiencies with the hydrophobic unnatural Ds base at the 5′‐end of the donor strand or at the 3′‐end of the acceptor strands. The rates of the ligation (turnover numbers) were calculated from the yields of the ligated products at 5 minutes (dsDNA: 0.5 μM; T4 DNA ligase: 0.0025 Weiss U/μL, 3.6 ± 0.9 nM) by averaging independent repetitive data (n = 2‐4, Figures 3 , S8 and S10 ), and indicated with the standard deviations

    Techniques Used: Ligation

    Ligation of (A) 5′‐phosphorylated R18X (donor strand, X = Ds or A) to FAM‐L22 (acceptor strand) and (B) 5′‐ phosphorylated R17 (donor strand) to FAM‐L23X (acceptor strand, X = Ds or A) in the presence of Template 25 (Y = Pa, T, C, A, or G). Reaction conditions: 0.05 Weiss U/μL T4 DNA ligase (corresponding to 1/5 of the amounts of ligase used in Figures 2 and 3 ), 0.5 μM each DNA, 10 minutes at 22 °C. The FAM‐labeled DNA bands in the gel were detected with an LAS4000 bio‐imager. The ligated products are 40‐mers. The DNA species higher than 40‐mer are expected to be nontemplate ligated products (with one extra donor strand, since we did not block the 3′‐end)
    Figure Legend Snippet: Ligation of (A) 5′‐phosphorylated R18X (donor strand, X = Ds or A) to FAM‐L22 (acceptor strand) and (B) 5′‐ phosphorylated R17 (donor strand) to FAM‐L23X (acceptor strand, X = Ds or A) in the presence of Template 25 (Y = Pa, T, C, A, or G). Reaction conditions: 0.05 Weiss U/μL T4 DNA ligase (corresponding to 1/5 of the amounts of ligase used in Figures 2 and 3 ), 0.5 μM each DNA, 10 minutes at 22 °C. The FAM‐labeled DNA bands in the gel were detected with an LAS4000 bio‐imager. The ligated products are 40‐mers. The DNA species higher than 40‐mer are expected to be nontemplate ligated products (with one extra donor strand, since we did not block the 3′‐end)

    Techniques Used: Ligation, Labeling, Blocking Assay

    Time course of the ligation reactions. A, Ligation of 5′‐phosphorylated R18X (donor strand) to FAM‐L22 (acceptor strand) in the presence of Template 25. B, Ligation of 5′‐phosphorylated R17 (donor strand) to FAM‐L23X (acceptor strand) in the presence of Template 25. X = Ds or A; Y = Pa, T, C, A, or G. Reaction conditions: 0.0025 Weiss U/μL T4 DNA ligase, 0.5 μM of each DNA at 22 °C. The time course of the reactions with the various base pairs were assessed by performing the ligation reactions at various time intervals: 1.25, 2.5, 5, 10 and 20 minutes
    Figure Legend Snippet: Time course of the ligation reactions. A, Ligation of 5′‐phosphorylated R18X (donor strand) to FAM‐L22 (acceptor strand) in the presence of Template 25. B, Ligation of 5′‐phosphorylated R17 (donor strand) to FAM‐L23X (acceptor strand) in the presence of Template 25. X = Ds or A; Y = Pa, T, C, A, or G. Reaction conditions: 0.0025 Weiss U/μL T4 DNA ligase, 0.5 μM of each DNA at 22 °C. The time course of the reactions with the various base pairs were assessed by performing the ligation reactions at various time intervals: 1.25, 2.5, 5, 10 and 20 minutes

    Techniques Used: Ligation

    Ligation of 5′‐phosphorylated R23 (A) or R22 (B) (donor strand) to L23X (A) or L24XT (B) (acceptor strand) in the presence of Template 35 (template strand). A 17‐mer DNA (17‐guide) was added to ensure the sufficient length of the duplex formation for T4 DNA ligase recognition. Reaction conditions: 0.25 Weiss U/μL T4 DNA ligase, 0.5 μM of each DNA fragment, 10 minutes at 22 °C. X = Ds or A, Y = Pa, T, C, A, or G. The DNA species higher than 46‐mer and those between 35‐mer and 46‐mer are expected to be nontemplate ligated products (with one extra donor strand, since we did not block the 3′ end)
    Figure Legend Snippet: Ligation of 5′‐phosphorylated R23 (A) or R22 (B) (donor strand) to L23X (A) or L24XT (B) (acceptor strand) in the presence of Template 35 (template strand). A 17‐mer DNA (17‐guide) was added to ensure the sufficient length of the duplex formation for T4 DNA ligase recognition. Reaction conditions: 0.25 Weiss U/μL T4 DNA ligase, 0.5 μM of each DNA fragment, 10 minutes at 22 °C. X = Ds or A, Y = Pa, T, C, A, or G. The DNA species higher than 46‐mer and those between 35‐mer and 46‐mer are expected to be nontemplate ligated products (with one extra donor strand, since we did not block the 3′ end)

    Techniques Used: Ligation, Blocking Assay

    Enzymatic ligation by T4 DNA ligase using DNA with hydrophobic UBPs. A, T4 DNA ligase domain organization and the schematic representation of enzymatic ligation. The N‐terminal domain (NTD, pink) corresponds to the DNA‐binding domain (DBD). The nucleotidyl transferase domain (NTase, yellow) contains the catalytic core, and OBD (orange) is the oligonucleotide‐binding domain. The DNA duplex sequences and the complex structure of T4 DNA ligase with the DNA duplex, containing the adenylated DNA intermediate (AppDNA), were adopted from PDB: 6DT1. B, Enzymatic ligation reactions, where the UBP (X‐Y; e.g., Ds‐Pa) is located at the ligation junction (nick site). Chemical structures of A‐T and A‐F (C), Ds‐Pa and Ds‐Px (D), and A‐Pa (E). The minor groove hydrogen bond acceptor residues are indicated in solid circles (pink). The 2‐fluorine residue, which has lower hydrogen‐bond acceptor ability, is indicated in an open circle (pink)
    Figure Legend Snippet: Enzymatic ligation by T4 DNA ligase using DNA with hydrophobic UBPs. A, T4 DNA ligase domain organization and the schematic representation of enzymatic ligation. The N‐terminal domain (NTD, pink) corresponds to the DNA‐binding domain (DBD). The nucleotidyl transferase domain (NTase, yellow) contains the catalytic core, and OBD (orange) is the oligonucleotide‐binding domain. The DNA duplex sequences and the complex structure of T4 DNA ligase with the DNA duplex, containing the adenylated DNA intermediate (AppDNA), were adopted from PDB: 6DT1. B, Enzymatic ligation reactions, where the UBP (X‐Y; e.g., Ds‐Pa) is located at the ligation junction (nick site). Chemical structures of A‐T and A‐F (C), Ds‐Pa and Ds‐Px (D), and A‐Pa (E). The minor groove hydrogen bond acceptor residues are indicated in solid circles (pink). The 2‐fluorine residue, which has lower hydrogen‐bond acceptor ability, is indicated in an open circle (pink)

    Techniques Used: Ligation, Binding Assay

    6) 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

    7) 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

    8) 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

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039251

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

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

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

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

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

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

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

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

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

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

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2019.4355

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

    Techniques Used: Plasmid Preparation, Construct

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx033

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

    Techniques Used: DNA Ligation, Ligation, Staining, Marker

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

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

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

    Techniques Used: Ligation, Sequencing, Ancient DNA Assay

    12) Product Images from "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

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

    Article Title: Practical Synthesis of Cap‐4 RNA

    Journal: Chembiochem

    doi: 10.1002/cbic.201900590

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

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

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

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

    Journal: Iranian Journal of Pharmaceutical Research : IJPR

    doi:

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

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

    15) 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

    16) 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

    17) 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

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

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

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-7-49

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

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

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

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

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

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

    Journal: Nature methods

    doi: 10.1038/nmeth.3742

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

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

    20) 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

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

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

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-7-49

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

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

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

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

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr860

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

    Techniques Used: Selection, Ligation, Polyacrylamide Gel Electrophoresis

    23) 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

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

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

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2019.4355

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

    Techniques Used: Plasmid Preparation, Construct

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

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

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2019.4355

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

    Techniques Used: Plasmid Preparation, Construct

    26) Product Images from "Structure of the Herpes Simplex Virus 1 Genome: Manipulation of Nicks and Gaps Can Abrogate Infectivity and Alter the Cellular DNA Damage Response"

    Article Title: Structure of the Herpes Simplex Virus 1 Genome: Manipulation of Nicks and Gaps Can Abrogate Infectivity and Alter the Cellular DNA Damage Response

    Journal: Journal of Virology

    doi: 10.1128/JVI.01723-14

    Incorporation of labeled nucleotides into HSV-DNA by Klenow fragment polymerase alone, Klenow and ligase together, or T4 DNA polymerase. Purified HSV-1 DNA was incubated with labeled nucleotides and the following enzymes, as described in Materials and
    Figure Legend Snippet: Incorporation of labeled nucleotides into HSV-DNA by Klenow fragment polymerase alone, Klenow and ligase together, or T4 DNA polymerase. Purified HSV-1 DNA was incubated with labeled nucleotides and the following enzymes, as described in Materials and

    Techniques Used: Labeling, Purification, Incubation

    27) 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

    28) 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

    29) 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

    30) 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

    31) 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

    32) 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

    33) Product Images from "TDP2-Dependent Non-Homologous End-Joining Protects against Topoisomerase II-Induced DNA Breaks and Genome Instability in Cells and In VivoTdp2: A Means to Fixing the Ends"

    Article Title: TDP2-Dependent Non-Homologous End-Joining Protects against Topoisomerase II-Induced DNA Breaks and Genome Instability in Cells and In VivoTdp2: A Means to Fixing the Ends

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003226

    Deletion of Tdp2 in mouse abolishes 5′-TDP activity and ligation of 5′ phosphotyrosine-blocked ends. A. Duplex substrate harbouring a 5′phosphotyrosine blunt end (left) was incubated with 9 µg Tdp2 +/+ or Tdp2 Δ1–3 tissue extract from bone marrow (BM), thymus and spleen for 1 h. B. Substrate in “A” was incubated with 1.5 µg of cellular extract from Tdp2 +/+ or Tdp2 Δ1–3 primary MEFs for the indicated time. C. Duplex substrate harbouring a 5′phosphotyrosine self-complementary overhang end (left) was incubated with 10 µg cellular extract from Tdp2 +/+ or Tdp2 Δ1–3 transformed MEFs for 2 h in the presence or absence of 50 mM EDTA. D. Self-ligation of 5′ phosphate (P) and 5′ phosphotyrosine (Y–P) overhang substrates as depicted in “C” incubated for 1.5 h with 3.3 µg cellular extract from Tdp2 +/+ or Tdp2 Δ1–3 transformed MEFs in the presence of T4 DNA ligase. In all cases migration of the 5′ phosphotyrosine substrate (Y–P), 5′ phosphate (P) and ligation (lig) products are indicated. E. Circularization efficiency of a linear plasmid with 5′ phosphotyrosine (Y–P) and 5′ phosphate (P) catalysed by Tdp2 Δ1–3 transformed MEFs extracts in the presence and absence of recombinant human TDP2 (hTDP2). Reaction products were transformed into E. coli and the number of transformants obtained per µg of initial substrate DNA (average ± s.e.m. of three independent experiments) is shown. Statistical significance by Two-way ANOVA test with Bonferroni post-test is indicated is shown.
    Figure Legend Snippet: Deletion of Tdp2 in mouse abolishes 5′-TDP activity and ligation of 5′ phosphotyrosine-blocked ends. A. Duplex substrate harbouring a 5′phosphotyrosine blunt end (left) was incubated with 9 µg Tdp2 +/+ or Tdp2 Δ1–3 tissue extract from bone marrow (BM), thymus and spleen for 1 h. B. Substrate in “A” was incubated with 1.5 µg of cellular extract from Tdp2 +/+ or Tdp2 Δ1–3 primary MEFs for the indicated time. C. Duplex substrate harbouring a 5′phosphotyrosine self-complementary overhang end (left) was incubated with 10 µg cellular extract from Tdp2 +/+ or Tdp2 Δ1–3 transformed MEFs for 2 h in the presence or absence of 50 mM EDTA. D. Self-ligation of 5′ phosphate (P) and 5′ phosphotyrosine (Y–P) overhang substrates as depicted in “C” incubated for 1.5 h with 3.3 µg cellular extract from Tdp2 +/+ or Tdp2 Δ1–3 transformed MEFs in the presence of T4 DNA ligase. In all cases migration of the 5′ phosphotyrosine substrate (Y–P), 5′ phosphate (P) and ligation (lig) products are indicated. E. Circularization efficiency of a linear plasmid with 5′ phosphotyrosine (Y–P) and 5′ phosphate (P) catalysed by Tdp2 Δ1–3 transformed MEFs extracts in the presence and absence of recombinant human TDP2 (hTDP2). Reaction products were transformed into E. coli and the number of transformants obtained per µg of initial substrate DNA (average ± s.e.m. of three independent experiments) is shown. Statistical significance by Two-way ANOVA test with Bonferroni post-test is indicated is shown.

    Techniques Used: Activity Assay, Ligation, Incubation, Transformation Assay, Migration, Plasmid Preparation, Recombinant

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1191

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

    Techniques Used: Incubation, Plasmid Preparation, Labeling

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

    Article Title: Practical Synthesis of Cap‐4 RNA

    Journal: Chembiochem

    doi: 10.1002/cbic.201900590

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

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

    36) 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

    37) 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

    38) Product Images from "A dumbbell probe-mediated rolling circle amplification strategy for highly sensitive microRNA detection"

    Article Title: A dumbbell probe-mediated rolling circle amplification strategy for highly sensitive microRNA detection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq556

    The D-RCA strategy for miRNA detection. A dumbbell probe contains three domains, an MBD, an SGBD and a loop domain. The binding of miRNA to MBD initiates RCA in the presence of T4 DNA ligase and phi29 polymerase, which generates a long DNA sequence that contains many SGBD for an amplified fluorescent readout.
    Figure Legend Snippet: The D-RCA strategy for miRNA detection. A dumbbell probe contains three domains, an MBD, an SGBD and a loop domain. The binding of miRNA to MBD initiates RCA in the presence of T4 DNA ligase and phi29 polymerase, which generates a long DNA sequence that contains many SGBD for an amplified fluorescent readout.

    Techniques Used: Binding Assay, Sequencing, Amplification

    The expression levels of hsa-miR-21 in total RNA samples with the dumbbell probe of ( a ) 50 nM and ( b ) 25 nM. Negative controls (−L) were performed with identical conditions in total RNA samples (+) without adding T4 DNA ligase. Statistical analysis was performed by paired t -test. Error bars (SD) were estimated from three independent measurements.
    Figure Legend Snippet: The expression levels of hsa-miR-21 in total RNA samples with the dumbbell probe of ( a ) 50 nM and ( b ) 25 nM. Negative controls (−L) were performed with identical conditions in total RNA samples (+) without adding T4 DNA ligase. Statistical analysis was performed by paired t -test. Error bars (SD) were estimated from three independent measurements.

    Techniques Used: Expressing

    39) Product Images from "Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells"

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158581

    Optimizations for second strand synthesis. (A) Schematic of the second strand synthesis procedure. Synthetic 5’ phosphorylated ODNs containing the lesion of interest are annealed to phagemid single-stranded DNA, complimentary strands are synthesised by T4 DNA polymerase, and ligated by T4 DNA ligase. (B) Second strand synthesis of HRAS construct using ssDNA purified by silica spin columns or anion-exchange columns. ssDNA purified by anion-exchange column produces high yields of covalently closed product. (C) Schematic of the alkaline gel analysis of the construct nicks positions. Double-digest of pcDNA3.1(+)-HRAS with SmaI and NdeI produces two fragments (labelled 1 and 2). If the synthetic ODN that becomes part of the transcribed strand is not ligated, the transcribed strand fragment 2 produces two smaller fragments (3 and 4). (D) Alkaline gel analysis of HRAS constructs. Negative control HRAS WT T5 exonuclease (T5 exo) treated, covalently closed construct produces only two bands and positive control Fpg nicked HRAS 8-oxoG constructs, treated and not treated with T5 exonuclease, produce the expected four bands. The anion-exchange purified HRAS WT construct produces only two bands, indicating the nicks following second strand synthesis occur at random positions.
    Figure Legend Snippet: Optimizations for second strand synthesis. (A) Schematic of the second strand synthesis procedure. Synthetic 5’ phosphorylated ODNs containing the lesion of interest are annealed to phagemid single-stranded DNA, complimentary strands are synthesised by T4 DNA polymerase, and ligated by T4 DNA ligase. (B) Second strand synthesis of HRAS construct using ssDNA purified by silica spin columns or anion-exchange columns. ssDNA purified by anion-exchange column produces high yields of covalently closed product. (C) Schematic of the alkaline gel analysis of the construct nicks positions. Double-digest of pcDNA3.1(+)-HRAS with SmaI and NdeI produces two fragments (labelled 1 and 2). If the synthetic ODN that becomes part of the transcribed strand is not ligated, the transcribed strand fragment 2 produces two smaller fragments (3 and 4). (D) Alkaline gel analysis of HRAS constructs. Negative control HRAS WT T5 exonuclease (T5 exo) treated, covalently closed construct produces only two bands and positive control Fpg nicked HRAS 8-oxoG constructs, treated and not treated with T5 exonuclease, produce the expected four bands. The anion-exchange purified HRAS WT construct produces only two bands, indicating the nicks following second strand synthesis occur at random positions.

    Techniques Used: Construct, Purification, Negative Control, Positive Control

    40) 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

    Related Articles

    Incubation:

    Article Title: Multiplex Single-Molecule DNA Barcoding Using an Oligonucleotide Ligation Assay
    Article Snippet: .. Immobilized target DNA was incubated with 50 nM of each upstream and 50 nM of each downstream barcode (independent of the number of different barcode sequences used) and 14 Weiss units/mL of T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA) in freshly prepared ligation buffer (40 mM Tris-HCl (pH 7.6), 10 mM MgCl2 , 10 mM dithiothreitol, 0.5 mM ATP) for 1 h at 25°C. .. Subsequently, the ligation buffer was replaced by imaging buffer (20 mM Tris-HCl (pH 8.0), 50 mM MgCl2 , 50 mM NaCl, 1 mM Trolox, 0.1 mg/mL glucose oxidase, 17 μ L/mL catalase, 0.8% (w/v) glucose) to enhance the photostability of the dyes during imaging ( ).

    Article Title: A novel single cell method to identify the genetic composition at a single nuclear body
    Article Snippet: Next, the cells were incubated for ten minutes in ISOL buffer (1X T4 DNA ligase reaction buffer (Fermentas) supplemented with 15% polyethylene glycol 8000 (PEG 8000), 0.5 mM adenosine triphosphate (ATP), and 0.05 mg/mL bovine serum albumin (BSA)). .. The cells were then incubated with ISOL buffer with the addition of 100 U/mL T4 DNA Ligase (Fermentas), and the appropriate oligo (hairpin oligo 35 μg/mL, double stranded oligo 0.29 nM). ..

    Article Title: Extract of Nippostrongylus brasiliensis Stimulates Polyclonal Type-2 Immunoglobulin Response by Inducing De Novo Class Switch
    Article Snippet: Each reaction mixture contained 10 μl of 10× React 3 buffer (1× final; Life Technologies), Eco RI (2 U/μg of DNA; Life Technologies), DNA solution, and the appropriate volume of double-distilled water required to adjust the volume to 100 μl. .. The reaction mixtures were then incubated overnight in a 37°C waterbath, following which the enzyme was inactivated by incubation at 70°C for 20 min. For ligation (circularization), 10 to 20 μl of digested DNA samples was placed in 1.5-ml microcentrifuge tubes to which 20 μl of 5× T4 DNA ligase buffer (1× final; Life Technologies), 2 μl (20 U) of T4 DNA ligase (Life Technologies), and double-distilled water were added to a final volume of 100 μl. .. The reaction mixtures were incubated overnight in a 16°C waterbath.

    Ligation:

    Article Title: Multiplex Single-Molecule DNA Barcoding Using an Oligonucleotide Ligation Assay
    Article Snippet: .. Immobilized target DNA was incubated with 50 nM of each upstream and 50 nM of each downstream barcode (independent of the number of different barcode sequences used) and 14 Weiss units/mL of T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA) in freshly prepared ligation buffer (40 mM Tris-HCl (pH 7.6), 10 mM MgCl2 , 10 mM dithiothreitol, 0.5 mM ATP) for 1 h at 25°C. .. Subsequently, the ligation buffer was replaced by imaging buffer (20 mM Tris-HCl (pH 8.0), 50 mM MgCl2 , 50 mM NaCl, 1 mM Trolox, 0.1 mg/mL glucose oxidase, 17 μ L/mL catalase, 0.8% (w/v) glucose) to enhance the photostability of the dyes during imaging ( ).

    Article Title: Extract of Nippostrongylus brasiliensis Stimulates Polyclonal Type-2 Immunoglobulin Response by Inducing De Novo Class Switch
    Article Snippet: Each reaction mixture contained 10 μl of 10× React 3 buffer (1× final; Life Technologies), Eco RI (2 U/μg of DNA; Life Technologies), DNA solution, and the appropriate volume of double-distilled water required to adjust the volume to 100 μl. .. The reaction mixtures were then incubated overnight in a 37°C waterbath, following which the enzyme was inactivated by incubation at 70°C for 20 min. For ligation (circularization), 10 to 20 μl of digested DNA samples was placed in 1.5-ml microcentrifuge tubes to which 20 μl of 5× T4 DNA ligase buffer (1× final; Life Technologies), 2 μl (20 U) of T4 DNA ligase (Life Technologies), and double-distilled water were added to a final volume of 100 μl. .. The reaction mixtures were incubated overnight in a 16°C waterbath.

    Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase
    Article Snippet: Reactions were incubated at 60°C for 1 h. For ligation reactions with T4 DNA ligase, adapter and splinter oligonucleotides were first hybridized by combining 200 pmol adapter (CL78, TL128, TL130 or TL134) and 400 pmol splinter (TL38, TL129, TL131 or TL135, respectively) in a 20 μl reaction containing 1 × T4 RNA ligase buffer (50 mM Tris-HCl, 10 mM NaCl, 1 mM DTT, pH 7.5 at 25°C; New England Biolabs) and heated up to 95°C for 10 s in a thermal cycler, followed by a ramp to 10°C at 0.1°C/s. .. 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). .. All ligation products were purified using Qiagen's Nucleotide Removal Kit according to the manufacturer's instructions but using MinElute columns (Qiagen) instead of Qiagen's QiaQuick columns to enable a reduction of the elution volume to 10 μl.

    Produced:

    Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase
    Article Snippet: .. CircLigase libraries with higher input volumes are also affected by other biases, including longer insert sizes, lower GC content and higher frequencies of C to T substitutions at their 3΄ end (Figure – ) than the ones produced by T4 DNA ligase. .. The pronounced asymmetry in C to T substitutions, which result from deamination of cytosine to uracil in ancient DNA , points to a less efficient ligation of 3΄ cytosines compared to thymines by CircLigase in the presence of high concentrations of DNA.

    Mass Spectrometry:

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain
    Article Snippet: This report showed that T4 PNK could not be detected in T4 DNA ligase. (PDF) Click here for additional data file. .. A report of MS analysis for T4 DNA ligase (Fermentas). .. This report showed that PNK could not be detected in T4 DNA ligase by using MS analysis. (PDF) Click here for additional data file.

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    Thermo Fisher t4 dna ligase
    DNA barcoding experimental scheme. Target DNA strands are immobilized on a microscope slide, and dye-labeled barcodes are introduced together with <t>T4</t> DNA ligase in the microfluidic chamber (1). Complementary barcodes bind transiently to the target site (2), whereas mismatched barcodes bind on an even shorter timescale (2′). Successful ligation is observed for the complementary barcodes (3) but not for the mismatched barcodes (3′). Ligation product shows stable binding to the target DNA (4), whereas mismatched barcodes dissociate and are washed away before imaging. To see this figure in color, go online.
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rlm 5 race
    Identification of siRNA-guided cleavage products of target mRNAs in rice. ( A ) mRNA 9639.m00201, ( B ) mRNA 9636.m02299 and ( C ) mRNA 9632.m00807. Mapping of cleavage sites was performed by <t>RLM-5′</t> <t>RACE.</t> Partial mRNA sequences from target genes were aligned with siRNAs. Numbers indicate the fraction of cloned PCR products terminating at different positions.
    Rlm 5 Race, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DNA barcoding experimental scheme. Target DNA strands are immobilized on a microscope slide, and dye-labeled barcodes are introduced together with T4 DNA ligase in the microfluidic chamber (1). Complementary barcodes bind transiently to the target site (2), whereas mismatched barcodes bind on an even shorter timescale (2′). Successful ligation is observed for the complementary barcodes (3) but not for the mismatched barcodes (3′). Ligation product shows stable binding to the target DNA (4), whereas mismatched barcodes dissociate and are washed away before imaging. To see this figure in color, go online.

    Journal: Biophysical Journal

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

    doi: 10.1016/j.bpj.2018.08.013

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

    Article Snippet: Immobilized target DNA was incubated with 50 nM of each upstream and 50 nM of each downstream barcode (independent of the number of different barcode sequences used) and 14 Weiss units/mL of T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA) in freshly prepared ligation buffer (40 mM Tris-HCl (pH 7.6), 10 mM MgCl2 , 10 mM dithiothreitol, 0.5 mM ATP) for 1 h at 25°C.

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

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

    Journal: Infection and Immunity

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

    doi:

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

    Article Snippet: The reaction mixtures were then incubated overnight in a 37°C waterbath, following which the enzyme was inactivated by incubation at 70°C for 20 min. For ligation (circularization), 10 to 20 μl of digested DNA samples was placed in 1.5-ml microcentrifuge tubes to which 20 μl of 5× T4 DNA ligase buffer (1× final; Life Technologies), 2 μl (20 U) of T4 DNA ligase (Life Technologies), and double-distilled water were added to a final volume of 100 μl.

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

    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

    Identification of siRNA-guided cleavage products of target mRNAs in rice. ( A ) mRNA 9639.m00201, ( B ) mRNA 9636.m02299 and ( C ) mRNA 9632.m00807. Mapping of cleavage sites was performed by RLM-5′ RACE. Partial mRNA sequences from target genes were aligned with siRNAs. Numbers indicate the fraction of cloned PCR products terminating at different positions.

    Journal: Nucleic Acids Research

    Article Title: Identification and characterization of endogenous small interfering RNAs from rice

    doi: 10.1093/nar/gki758

    Figure Lengend Snippet: Identification of siRNA-guided cleavage products of target mRNAs in rice. ( A ) mRNA 9639.m00201, ( B ) mRNA 9636.m02299 and ( C ) mRNA 9632.m00807. Mapping of cleavage sites was performed by RLM-5′ RACE. Partial mRNA sequences from target genes were aligned with siRNAs. Numbers indicate the fraction of cloned PCR products terminating at different positions.

    Article Snippet: RLM-5′ RACE (RNA ligase-mediated-5′ rapid amplification of cDNA ends) was carried out with use of the GeneRacer Kit (Invitrogen Life Technologies).

    Techniques: Clone Assay, Polymerase Chain Reaction