t4 dna ligase buffer  (Thermo Fisher)


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    Name:
    T4 DNA Ligase Buffer
    Description:
    T4 DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of ATP between double stranded DNAs with 3 hydroxyl and 5 phosphate termini The unique T4 DNA Ligase buffer optimizes ligation which can be performed in 5 minutes 1 Single stranded nucleic acids are not substrates for this enzyme A T4 DNA Ligase Technical Bulletin is available Applications Cloning blunt end or cohesive end ligation 2 Adding linkers or adapters to blunt ended DNA 2 Source Purified from E coli œ lysogen NM989 Performance and Quality Testing Endodeoxyribonuclease 3 and 5 exodeoxyribonuclease assays ligation efficiency tested Unit Definition One unit catalyzes the exchange of 1 nmol 32P labeled pyrophosphate into ATP in 20 min at 37°C One unit is equal to approximately 300 cohesive end ligation units Unit Reaction Conditions 66 mM Tris HCl pH 7 6 6 6 mM MgCl2 10 mM DTT 66 µM ATP 3 3 µM 32 P labeled pyrophosphate and enzyme in 0 1 ml for 20 min at 37°C
    Catalog Number:
    46300018
    Price:
    None
    Applications:
    ChIP-on-Chip|Cloning|RNAi, Epigenetics & Non-Coding RNA Research|Chromatin Biology|Restriction Enzyme Cloning
    Category:
    Lab Reagents and Chemicals
    Buy from Supplier


    Structured Review

    Thermo Fisher t4 dna ligase buffer
    Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard <t>T4</t> DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.
    T4 DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of ATP between double stranded DNAs with 3 hydroxyl and 5 phosphate termini The unique T4 DNA Ligase buffer optimizes ligation which can be performed in 5 minutes 1 Single stranded nucleic acids are not substrates for this enzyme A T4 DNA Ligase Technical Bulletin is available Applications Cloning blunt end or cohesive end ligation 2 Adding linkers or adapters to blunt ended DNA 2 Source Purified from E coli œ lysogen NM989 Performance and Quality Testing Endodeoxyribonuclease 3 and 5 exodeoxyribonuclease assays ligation efficiency tested Unit Definition One unit catalyzes the exchange of 1 nmol 32P labeled pyrophosphate into ATP in 20 min at 37°C One unit is equal to approximately 300 cohesive end ligation units Unit Reaction Conditions 66 mM Tris HCl pH 7 6 6 6 mM MgCl2 10 mM DTT 66 µM ATP 3 3 µM 32 P labeled pyrophosphate and enzyme in 0 1 ml for 20 min at 37°C
    https://www.bioz.com/result/t4 dna ligase buffer/product/Thermo Fisher
    Average 96 stars, based on 96 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase buffer - by Bioz Stars, 2020-07
    96/100 stars

    Images

    1) 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

    2) 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

    3) 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

    Related Articles

    Clone Assay:

    Article Title: Assembly of custom TALE-type DNA binding domains by modular cloning
    Article Snippet: .. Cut-ligation cloning protocol For cut-ligation reaction 40 fmol of each plasmid, ligation buffer (Fermentas), 15 U of either Bsa I or Bpi I and 15 U high-concentrated T4 DNA ligase were used in a 20 μl volume. .. One microliter reaction mix was added to 50 μl chemical competent TOP10 cells, incubated for 15 min on ice and transformed by heat shock.

    Incubation:

    Article Title: Extract of Nippostrongylus brasiliensis Stimulates Polyclonal Type-2 Immunoglobulin Response by Inducing De Novo Class Switch
    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. .. The reaction mixtures were incubated overnight in a 16°C waterbath.

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection
    Article Snippet: .. Equal molar amounts (typically 250–500 ng at ∼ 100 – 250 ng/µl ) of orthogonally protected synthons were mixed, 0.5–1 unit T4 ligase (Fermentas) and T4 ligase buffer (Fermentas) were added and the ligation mixture was incubated for 10–20 min at 16°C. .. Adding additional ligase had little effect on ligation efficiency.

    Article Title: Establishing broad generality of DNA catalysts for site-specific hydrolysis of single-stranded DNA
    Article Snippet: .. The sample was then brought to 20 µl total volume containing 1× of T4 DNA ligase buffer and one unit of T4 DNA ligase (Fermentas), incubated for 1 h at 37°C, and separated by 8% PAGE. ..

    Plasmid Preparation:

    Article Title: Assembly of custom TALE-type DNA binding domains by modular cloning
    Article Snippet: .. Cut-ligation cloning protocol For cut-ligation reaction 40 fmol of each plasmid, ligation buffer (Fermentas), 15 U of either Bsa I or Bpi I and 15 U high-concentrated T4 DNA ligase were used in a 20 μl volume. .. One microliter reaction mix was added to 50 μl chemical competent TOP10 cells, incubated for 15 min on ice and transformed by heat shock.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Establishing broad generality of DNA catalysts for site-specific hydrolysis of single-stranded DNA
    Article Snippet: .. The sample was then brought to 20 µl total volume containing 1× of T4 DNA ligase buffer and one unit of T4 DNA ligase (Fermentas), incubated for 1 h at 37°C, and separated by 8% PAGE. ..

    Ligation:

    Article Title: PARALLEL EVOLUTION OF LOCAL ADAPTATION AND REPRODUCTIVE ISOLATION IN THE FACE OF GENE FLOW
    Article Snippet: .. Then, 5.5 μL of 1× ligation buffer (Invitrogen, Carlsbad, CA) containing 0.5U of T4 DNA ligase (Invitrogen), 0.9 μM Eco-adaptor, and 0.9 μM Pst-adaptor were added to the digestion reaction. .. Ligation reactions were diluted 1:4 and used as template for preselective PCRs.

    Article Title: Dissolution of Xylose Metabolism in Lactococcus lactis
    Article Snippet: .. Ligation reactions contained restricted DNA at a series of concentrations from 25 to 750 ng, together with 1 U of T4 DNA ligase (Gibco, Gaithersburg, Md.), 10 μl of 5× ligation buffer (Gibco), and sterile distilled H2 O, for a final reaction volume of 50 μl. .. The resulting self-ligated DNA was then precipitated with NaCl and ethanol and then resuspended in sterile distilled H2 O to give a final concentration of 5 ng/μl for use in PCR.

    Article Title: Extract of Nippostrongylus brasiliensis Stimulates Polyclonal Type-2 Immunoglobulin Response by Inducing De Novo Class Switch
    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. .. The reaction mixtures were incubated overnight in a 16°C waterbath.

    Article Title: SPlinted Ligation Adapter Tagging (SPLAT), a novel library preparation method for whole genome bisulphite sequencing
    Article Snippet: .. For the 3΄-end ligation; adapter ss1 (final conc 10 μM), T4 DNA ligase buffer (40 mM Tris–HCl pH 7.8,10 mM MgCl2 , 10 mM DTT, 0.5 mM ATP), PEG4000 (5% w/v) and 30 units T4 DNA ligase (Thermo Fisher Scientific) and nuclease free water was added to the sample on ice, in a total volume of 30 μl. .. Ligation reaction mixtures were incubated at 20°C for 1 h, and subsequently purified using AMPure XP (BeckmanCoulter) beads in a 2:1 bead to sample ratio and eluted with 10 μl nuclease free water.

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection
    Article Snippet: .. Equal molar amounts (typically 250–500 ng at ∼ 100 – 250 ng/µl ) of orthogonally protected synthons were mixed, 0.5–1 unit T4 ligase (Fermentas) and T4 ligase buffer (Fermentas) were added and the ligation mixture was incubated for 10–20 min at 16°C. .. Adding additional ligase had little effect on ligation efficiency.

    Article Title: Assembly of custom TALE-type DNA binding domains by modular cloning
    Article Snippet: .. Cut-ligation cloning protocol For cut-ligation reaction 40 fmol of each plasmid, ligation buffer (Fermentas), 15 U of either Bsa I or Bpi I and 15 U high-concentrated T4 DNA ligase were used in a 20 μl volume. .. One microliter reaction mix was added to 50 μl chemical competent TOP10 cells, incubated for 15 min on ice and transformed by heat shock.

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    Thermo Fisher t4 dna ligase buffer
    Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard <t>T4</t> DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.
    T4 Dna Ligase Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase buffer/product/Thermo Fisher
    Average 96 stars, based on 96 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase buffer - by Bioz Stars, 2020-07
    96/100 stars
      Buy from Supplier

    94
    Thermo Fisher t4 dna ligation buffer
    Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard <t>T4</t> DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.
    T4 Dna Ligation Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligation buffer/product/Thermo Fisher
    Average 94 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligation buffer - by Bioz Stars, 2020-07
    94/100 stars
      Buy from Supplier

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkw1110

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

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

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

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkw1110

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

    Article Snippet: For the 5΄-end ligation; ss2 (final conc 10 μM), T4 DNA ligation buffer, PEG4000 (5%, w/v) and 30 units T4 DNA ligase (Thermo Fisher Scientific) and nuclease free H2 O was added to the sample on ice, in a total volume of 20 μl.

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