t4 dna polymerase Thermo Fisher Search Results


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  • 99
    New England Biolabs t4 pnk
    T4 Pnk, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher thermo fisher scientific t4 polymerase
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
    Thermo Fisher Scientific T4 Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 216 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher t4 dna polymerase
    Single-stranded DNA ligation with <t>T4</t> 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.
    T4 Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 2083 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dna polymerase dnase i
    Single-stranded DNA ligation with <t>T4</t> 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.
    Dna Polymerase Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche dna polymerase buffer
    Single-stranded DNA ligation with <t>T4</t> 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.
    Dna Polymerase Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher 1x t4 dna polymerase buffer
    Single-stranded DNA ligation with <t>T4</t> 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.
    1x T4 Dna Polymerase Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pfu dna polymerases
    Single-stranded DNA ligation with <t>T4</t> 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.
    Pfu Dna Polymerases, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dna polymerase i dnase i
    Single-stranded DNA ligation with <t>T4</t> 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.
    Dna Polymerase I Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 18427 013 klenow dna polymerase t4 pnk minelute reaction cleanup kit
    Single-stranded DNA ligation with <t>T4</t> 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.
    18427 013 Klenow Dna Polymerase T4 Pnk Minelute Reaction Cleanup Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher hindiii
    Single-stranded DNA ligation with <t>T4</t> 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.
    Hindiii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6951 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pyes2
    Single-stranded DNA ligation with <t>T4</t> 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.
    Pyes2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 2098 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher apai
    Single-stranded DNA ligation with <t>T4</t> 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.
    Apai, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 683 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ecori
    Single-stranded DNA ligation with <t>T4</t> 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.
    Ecori, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9775 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher t4 ligase buffer
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
    T4 Ligase Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 246 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher u t4 ligase
    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 <t>T4</t> DNA ligase to construct the final FSH1 double stranded RNA interference plasmid pCB309-PFUFT. FSH1, family of serine hydrolases 1.
    U T4 Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 481 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher second strand synthesis
    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 <t>T4</t> DNA ligase to construct the final FSH1 double stranded RNA interference plasmid pCB309-PFUFT. FSH1, family of serine hydrolases 1.
    Second Strand Synthesis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fisher s t4 ligase enzyme
    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 <t>T4</t> DNA ligase to construct the final FSH1 double stranded RNA interference plasmid pCB309-PFUFT. FSH1, family of serine hydrolases 1.
    Fisher S T4 Ligase Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher t4 polynucleotide kinase
    Workflow of PEP. Double-stranded template DNA is blunt-end repaired using T4 DNA polymerase and <t>T4</t> polynucleotide kinase (not depicted). ( I ) Using T4 DNA ligase, biotinylated adapters are attached to both ends of the template molecules. The blunt end ligation reaction also produces adapter dimers, which are subsequently removed by size selective purification. ( II ) 5′-tailed primers carrying the 454 ‘B’ sequence (shown in blue) are hybridized to the overhanging 3′-ends of the adapters. Primer extension is carried out under reaction conditions optimal for the assayed polymerase. Unless second-strand synthesis stops prematurely, due to a blocking lesion, a nick or random polymerase stalling, the flanking adapter sequence (shown in red) is copied. ( III ) Primer extension products are captured on streptavidine beads to remove excess primers and extension products from nicked template strands. Extension products are released by heat denaturation. ( IV ) A 454 sequencing library is created by attaching single-stranded adapters with the 454 ‘A’ sequence (shown in green) to the 3′-ends. The sequencing library is converted to double-stranded form (not depicted) to allow for efficient removal of excess A-adapters. The 454 sequencing is initiated from the A-adapter. If primer extensions were complete, sequences will start with an 8-bp adapter sequence, which serves as the end-of-template recognition sequence (framed by rectangles).
    T4 Polynucleotide Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7878 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t4 dna ligase
    Workflow of PEP. Double-stranded template DNA is blunt-end repaired using T4 DNA polymerase and <t>T4</t> polynucleotide kinase (not depicted). ( I ) Using T4 DNA ligase, biotinylated adapters are attached to both ends of the template molecules. The blunt end ligation reaction also produces adapter dimers, which are subsequently removed by size selective purification. ( II ) 5′-tailed primers carrying the 454 ‘B’ sequence (shown in blue) are hybridized to the overhanging 3′-ends of the adapters. Primer extension is carried out under reaction conditions optimal for the assayed polymerase. Unless second-strand synthesis stops prematurely, due to a blocking lesion, a nick or random polymerase stalling, the flanking adapter sequence (shown in red) is copied. ( III ) Primer extension products are captured on streptavidine beads to remove excess primers and extension products from nicked template strands. Extension products are released by heat denaturation. ( IV ) A 454 sequencing library is created by attaching single-stranded adapters with the 454 ‘A’ sequence (shown in green) to the 3′-ends. The sequencing library is converted to double-stranded form (not depicted) to allow for efficient removal of excess A-adapters. The 454 sequencing is initiated from the A-adapter. If primer extensions were complete, sequences will start with an 8-bp adapter sequence, which serves as the end-of-template recognition sequence (framed by rectangles).
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 38631 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cut pselect gfpzeo
    Workflow of PEP. Double-stranded template DNA is blunt-end repaired using T4 DNA polymerase and <t>T4</t> polynucleotide kinase (not depicted). ( I ) Using T4 DNA ligase, biotinylated adapters are attached to both ends of the template molecules. The blunt end ligation reaction also produces adapter dimers, which are subsequently removed by size selective purification. ( II ) 5′-tailed primers carrying the 454 ‘B’ sequence (shown in blue) are hybridized to the overhanging 3′-ends of the adapters. Primer extension is carried out under reaction conditions optimal for the assayed polymerase. Unless second-strand synthesis stops prematurely, due to a blocking lesion, a nick or random polymerase stalling, the flanking adapter sequence (shown in red) is copied. ( III ) Primer extension products are captured on streptavidine beads to remove excess primers and extension products from nicked template strands. Extension products are released by heat denaturation. ( IV ) A 454 sequencing library is created by attaching single-stranded adapters with the 454 ‘A’ sequence (shown in green) to the 3′-ends. The sequencing library is converted to double-stranded form (not depicted) to allow for efficient removal of excess A-adapters. The 454 sequencing is initiated from the A-adapter. If primer extensions were complete, sequences will start with an 8-bp adapter sequence, which serves as the end-of-template recognition sequence (framed by rectangles).
    Cut Pselect Gfpzeo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher hc t4 ligase
    Workflow of PEP. Double-stranded template DNA is blunt-end repaired using T4 DNA polymerase and <t>T4</t> polynucleotide kinase (not depicted). ( I ) Using T4 DNA ligase, biotinylated adapters are attached to both ends of the template molecules. The blunt end ligation reaction also produces adapter dimers, which are subsequently removed by size selective purification. ( II ) 5′-tailed primers carrying the 454 ‘B’ sequence (shown in blue) are hybridized to the overhanging 3′-ends of the adapters. Primer extension is carried out under reaction conditions optimal for the assayed polymerase. Unless second-strand synthesis stops prematurely, due to a blocking lesion, a nick or random polymerase stalling, the flanking adapter sequence (shown in red) is copied. ( III ) Primer extension products are captured on streptavidine beads to remove excess primers and extension products from nicked template strands. Extension products are released by heat denaturation. ( IV ) A 454 sequencing library is created by attaching single-stranded adapters with the 454 ‘A’ sequence (shown in green) to the 3′-ends. The sequencing library is converted to double-stranded form (not depicted) to allow for efficient removal of excess A-adapters. The 454 sequencing is initiated from the A-adapter. If primer extensions were complete, sequences will start with an 8-bp adapter sequence, which serves as the end-of-template recognition sequence (framed by rectangles).
    Hc T4 Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher t4 dna ligase reaction 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 Reaction Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anza t4 dna ligase master mix
    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.
    Anza T4 Dna Ligase Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phusiontm site directed mutagenesis kit
    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.
    Phusiontm Site Directed Mutagenesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher t4 dna polimerase
    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.
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    Thermo Fisher t4 ligase rapid kit
    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.
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    Thermo Fisher ambion t4 rna ligase
    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.
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    Thermo Fisher buffers invitrogen t4 ligase
    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.
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    Thermo Fisher dna manipulation
    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.
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    Thermo Fisher t4 ligase kit
    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.
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    Image Search Results


    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.

    Journal: PLoS ONE

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection

    doi: 10.1371/journal.pone.0037617

    Figure Lengend Snippet: Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.

    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.

    Techniques: Incubation, Plasmid Preparation, Clone Assay, Expressing, Construct

    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: For fill-in with T4 DNA polymerase a 50 μl reaction mix was prepared containing 1× T4 DNA polymerase buffer (ThermoFisher Scientific), 0.05% Tween-20, 100 μM each dNTP, 100 pmol primer CL130 and 2 μl 5 U/μl T4 DNA polymerase (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: For fill-in with T4 DNA polymerase a 50 μl reaction mix was prepared containing 1× T4 DNA polymerase buffer (ThermoFisher Scientific), 0.05% Tween-20, 100 μM each dNTP, 100 pmol primer CL130 and 2 μl 5 U/μl T4 DNA polymerase (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: For fill-in with T4 DNA polymerase a 50 μl reaction mix was prepared containing 1× T4 DNA polymerase buffer (ThermoFisher Scientific), 0.05% Tween-20, 100 μM each dNTP, 100 pmol primer CL130 and 2 μl 5 U/μl T4 DNA polymerase (ThermoFisher Scientific).

    Techniques: Ligation, Sequencing, Ancient DNA Assay

    UV damage does not affect NCP reconstitution with the 601 sequence. A , NCP reconstitution with UV-undamaged and -damaged DNA. The 147-bp 601 DNA containing both UV lesions and labeling were mixed with histone octamer at 2 m NaCl. The reconstitution was performed by stepwise salt dialysis, and the final NaCl concentration was 50 m m . The reconstituted products were resolved in 5% native polyacrylamide gel and stained with SYBR Gold. The 100-bp DNA markers are indicated on the left. B , presence of CPDs and 6-4PPs in UV-damaged DNA. The different UV-damaged DNA were blotted on the nitrocellulose and detected by lesion-specific antibodies. The same membranes were reprobed with 32 P-labeled DNA to show equal loading. C , Southern blot of the photoproduct yield of the UV-irradiated DNA fragment. The DNA was treated with or without photolyase prior to the T4 DNA polymerase ( pol ) digestion. The digested samples were blotted on the nylon membrane and probed with with 32 P-labeled DNA. D , quantification data of the photoproduct yield by Southern blots. The CPD signals were calculated by subtracting the total signals with the 6-4PPs signals. Three independent experiments were performed to show error bars .

    Journal: The Journal of Biological Chemistry

    Article Title: UV Damage in DNA Promotes Nucleosome Unwrapping *

    doi: 10.1074/jbc.M110.140087

    Figure Lengend Snippet: UV damage does not affect NCP reconstitution with the 601 sequence. A , NCP reconstitution with UV-undamaged and -damaged DNA. The 147-bp 601 DNA containing both UV lesions and labeling were mixed with histone octamer at 2 m NaCl. The reconstitution was performed by stepwise salt dialysis, and the final NaCl concentration was 50 m m . The reconstituted products were resolved in 5% native polyacrylamide gel and stained with SYBR Gold. The 100-bp DNA markers are indicated on the left. B , presence of CPDs and 6-4PPs in UV-damaged DNA. The different UV-damaged DNA were blotted on the nitrocellulose and detected by lesion-specific antibodies. The same membranes were reprobed with 32 P-labeled DNA to show equal loading. C , Southern blot of the photoproduct yield of the UV-irradiated DNA fragment. The DNA was treated with or without photolyase prior to the T4 DNA polymerase ( pol ) digestion. The digested samples were blotted on the nylon membrane and probed with with 32 P-labeled DNA. D , quantification data of the photoproduct yield by Southern blots. The CPD signals were calculated by subtracting the total signals with the 6-4PPs signals. Three independent experiments were performed to show error bars .

    Article Snippet: Briefly, samples were incubated with 2.5 units of T4 DNA polymerase-exonuclease (Fermentas) at 37 °C for 2 h. The reaction was stopped by heating at 65 °C for 10 min.

    Techniques: Sequencing, Labeling, Concentration Assay, Staining, Southern Blot, Irradiation

    J κ 1 and J H 2 coding ends have 3′ overhangs. (A and B) DNA purified in agarose plugs from newborn-mouse thymocytes (A) or 103 bcl2/4 cells cultured at 33 or 39°C (B) (shift − or +) was ligated to the BW linker without pretreatment (lanes none) or after treatment with either T4 DNA polymerase (lanes T4 Pol) or mung bean nuclease (lanes MB-N). Ligated plugs were analyzed by PCR for J H 2 (A) or J κ 1 (B) coding-end (ce) breaks (arrows). The lanes labeled 63-12 were control LM-PCR assays with 63-12 cell DNA. Lanes 1 and 2 in panel A represent independent thymocyte DNA samples. (C) Amplified products from lanes 1, 3, and 4 in panel A and lanes 2, 4, and 6 in panel B were gel purified, reamplified for five cycles with a 32 P-labeled specific oligonucleotide, and analyzed by denaturing polyacrylamide gel electrophoresis. In each case, the arrow indicates the predominant broken-ended molecule (+9 for J H 2 and +4 for J κ 1) and the tick marks indicate 1-nt intervals (determined by the electrophoresis of a DNA sequencing reaction mixture on the same gel). Lanes: N, no pretreatment; T, T4 DNA polymerase pretreatment; M, mung bean nuclease pretreatment.

    Journal: Molecular and Cellular Biology

    Article Title: Structure of Nonhairpin Coding-End DNA Breaks in Cells Undergoing V(D)J Recombination

    doi:

    Figure Lengend Snippet: J κ 1 and J H 2 coding ends have 3′ overhangs. (A and B) DNA purified in agarose plugs from newborn-mouse thymocytes (A) or 103 bcl2/4 cells cultured at 33 or 39°C (B) (shift − or +) was ligated to the BW linker without pretreatment (lanes none) or after treatment with either T4 DNA polymerase (lanes T4 Pol) or mung bean nuclease (lanes MB-N). Ligated plugs were analyzed by PCR for J H 2 (A) or J κ 1 (B) coding-end (ce) breaks (arrows). The lanes labeled 63-12 were control LM-PCR assays with 63-12 cell DNA. Lanes 1 and 2 in panel A represent independent thymocyte DNA samples. (C) Amplified products from lanes 1, 3, and 4 in panel A and lanes 2, 4, and 6 in panel B were gel purified, reamplified for five cycles with a 32 P-labeled specific oligonucleotide, and analyzed by denaturing polyacrylamide gel electrophoresis. In each case, the arrow indicates the predominant broken-ended molecule (+9 for J H 2 and +4 for J κ 1) and the tick marks indicate 1-nt intervals (determined by the electrophoresis of a DNA sequencing reaction mixture on the same gel). Lanes: N, no pretreatment; T, T4 DNA polymerase pretreatment; M, mung bean nuclease pretreatment.

    Article Snippet: Some plug DNA samples were subjected to T4 DNA polymerase treatment by incubating 40 μl of plug in an 80-μl reaction mixture with manufacturer’s buffer (Life Technologies), 5 U of T4 DNA polymerase, and 100 μM deoxynucleoside triphosphates at 37°C for 1 h. Other plugs were treated with various amounts of mung bean nuclease (BRL) under conditions previously reported by Zhu and Roth ( ).

    Techniques: Purification, Cell Culture, Polymerase Chain Reaction, Labeling, Amplification, Polyacrylamide Gel Electrophoresis, Electrophoresis, DNA Sequencing

    V(D)J recombination reaction pathway and LM-PCR assay for reaction intermediates. (A) Diagram of the reactants (top), broken DNA intermediates (middle), and products (bottom) of V(D)J recombination. V and J gene segments, with their associated RSSs (heptamer [H] and nonamer [N]) are recognized and cleaved by the recombinase at the RSS–coding-segment junction (arrow), generating coding-end and signal-end fragments. These ends are joined to form a coding joint and a signal joint. (B) LM-PCR assay for broken-ended recombination reaction intermediates. The BW linker is ligated to available ends in total genomic DNA by using T4 DNA ligase. The sites of linker ligation are revealed by a set of nested PCR assays with a linker primer (BW-1) and locus-specific primers (open arrows labeled 1, 2, 4, and 5). Blots of PCR products were probed with internal oligonucleotides (solid lines labeled 3 and 6).

    Journal: Molecular and Cellular Biology

    Article Title: Structure of Nonhairpin Coding-End DNA Breaks in Cells Undergoing V(D)J Recombination

    doi:

    Figure Lengend Snippet: V(D)J recombination reaction pathway and LM-PCR assay for reaction intermediates. (A) Diagram of the reactants (top), broken DNA intermediates (middle), and products (bottom) of V(D)J recombination. V and J gene segments, with their associated RSSs (heptamer [H] and nonamer [N]) are recognized and cleaved by the recombinase at the RSS–coding-segment junction (arrow), generating coding-end and signal-end fragments. These ends are joined to form a coding joint and a signal joint. (B) LM-PCR assay for broken-ended recombination reaction intermediates. The BW linker is ligated to available ends in total genomic DNA by using T4 DNA ligase. The sites of linker ligation are revealed by a set of nested PCR assays with a linker primer (BW-1) and locus-specific primers (open arrows labeled 1, 2, 4, and 5). Blots of PCR products were probed with internal oligonucleotides (solid lines labeled 3 and 6).

    Article Snippet: Some plug DNA samples were subjected to T4 DNA polymerase treatment by incubating 40 μl of plug in an 80-μl reaction mixture with manufacturer’s buffer (Life Technologies), 5 U of T4 DNA polymerase, and 100 μM deoxynucleoside triphosphates at 37°C for 1 h. Other plugs were treated with various amounts of mung bean nuclease (BRL) under conditions previously reported by Zhu and Roth ( ).

    Techniques: Polymerase Chain Reaction, Ligation, Nested PCR, Labeling

    T4 DNA polymerase treatment enhances LM-PCR detection of broken coding ends. (A to C) DNA samples prepared by the agarose plug method from uninduced (33°C [33 degr]) and induced (39°C [39 degr]) 103 bcl2/4 cells (A and B) and from newborn thymus (C) were analyzed by LM-PCR for broken J κ 1 coding (A), J κ 1 and J κ 2 signal (B), and J H 2 coding and signal (C) ends without (lanes −) or with (lanes +) T4 DNA polymerase (T4 pol) pretreatment. Controls included identically prepared and treated 63-12 (RAG-2-deficient) cell DNA and buffer (lane C). DNA samples from panel C were amplified with primers specific for a nonrearranging genomic locus, demonstrating the presence of DNA in all samples. (D) Ethidium bromide-stained agarose gel of these control amplifications. Lanes 1 to 4 correspond to samples 1 to 4 in panel C, lanes 5 to 8 correspond to samples 6 to 9 in panel C, and lane 9 is a buffer-only control amplification. Lanes 1 to 6 of panels A and B were shown to contain equivalent amounts of DNA by a similar method (data not shown). ce, coding ends; se, signal ends.

    Journal: Molecular and Cellular Biology

    Article Title: Structure of Nonhairpin Coding-End DNA Breaks in Cells Undergoing V(D)J Recombination

    doi:

    Figure Lengend Snippet: T4 DNA polymerase treatment enhances LM-PCR detection of broken coding ends. (A to C) DNA samples prepared by the agarose plug method from uninduced (33°C [33 degr]) and induced (39°C [39 degr]) 103 bcl2/4 cells (A and B) and from newborn thymus (C) were analyzed by LM-PCR for broken J κ 1 coding (A), J κ 1 and J κ 2 signal (B), and J H 2 coding and signal (C) ends without (lanes −) or with (lanes +) T4 DNA polymerase (T4 pol) pretreatment. Controls included identically prepared and treated 63-12 (RAG-2-deficient) cell DNA and buffer (lane C). DNA samples from panel C were amplified with primers specific for a nonrearranging genomic locus, demonstrating the presence of DNA in all samples. (D) Ethidium bromide-stained agarose gel of these control amplifications. Lanes 1 to 4 correspond to samples 1 to 4 in panel C, lanes 5 to 8 correspond to samples 6 to 9 in panel C, and lane 9 is a buffer-only control amplification. Lanes 1 to 6 of panels A and B were shown to contain equivalent amounts of DNA by a similar method (data not shown). ce, coding ends; se, signal ends.

    Article Snippet: Some plug DNA samples were subjected to T4 DNA polymerase treatment by incubating 40 μl of plug in an 80-μl reaction mixture with manufacturer’s buffer (Life Technologies), 5 U of T4 DNA polymerase, and 100 μM deoxynucleoside triphosphates at 37°C for 1 h. Other plugs were treated with various amounts of mung bean nuclease (BRL) under conditions previously reported by Zhu and Roth ( ).

    Techniques: Polymerase Chain Reaction, Amplification, Staining, Agarose Gel Electrophoresis

    Length heterogeneity of amplified coding ends and joints. D H , J H 1, J H 2, V κ , and J κ 1 coding ends (ce) (A, B, C, E, and F) and DJ H and VJ κ joints (D and G) were amplified from T4 polymerase-treated thymus or induced 103 bcl2/4 cell DNA. The amplified fragments were gel purified and end labeled with [γ- 32 P]ATP by using T4 DNA kinase. Labeled fragments were electrophoresed on 6% denaturing polyacrylamide gels alongside DNA sequencing ladders used as size markers. The diagrams adjacent to each coding-fragment gel image indicate the sequence position based on these comigrating sequence markers. Position zero in the diagrams corresponds to the full-length coding end (i.e., the junction between the RSS and the coding segment), positive numbers indicate coding-end deletions, and negative numbers indicate longer-than-full-length coding-end lengths. The diagrams adjoining DJ H and VJ κ joints indicate successive nucleotide lengths. In panel E, the lane labeled Vκ mkr contains a radiolabeled amplification product of genomic DNA demonstrating the length heterogeneity of intact V κ genes (see the text).

    Journal: Molecular and Cellular Biology

    Article Title: Structure of Nonhairpin Coding-End DNA Breaks in Cells Undergoing V(D)J Recombination

    doi:

    Figure Lengend Snippet: Length heterogeneity of amplified coding ends and joints. D H , J H 1, J H 2, V κ , and J κ 1 coding ends (ce) (A, B, C, E, and F) and DJ H and VJ κ joints (D and G) were amplified from T4 polymerase-treated thymus or induced 103 bcl2/4 cell DNA. The amplified fragments were gel purified and end labeled with [γ- 32 P]ATP by using T4 DNA kinase. Labeled fragments were electrophoresed on 6% denaturing polyacrylamide gels alongside DNA sequencing ladders used as size markers. The diagrams adjacent to each coding-fragment gel image indicate the sequence position based on these comigrating sequence markers. Position zero in the diagrams corresponds to the full-length coding end (i.e., the junction between the RSS and the coding segment), positive numbers indicate coding-end deletions, and negative numbers indicate longer-than-full-length coding-end lengths. The diagrams adjoining DJ H and VJ κ joints indicate successive nucleotide lengths. In panel E, the lane labeled Vκ mkr contains a radiolabeled amplification product of genomic DNA demonstrating the length heterogeneity of intact V κ genes (see the text).

    Article Snippet: Some plug DNA samples were subjected to T4 DNA polymerase treatment by incubating 40 μl of plug in an 80-μl reaction mixture with manufacturer’s buffer (Life Technologies), 5 U of T4 DNA polymerase, and 100 μM deoxynucleoside triphosphates at 37°C for 1 h. Other plugs were treated with various amounts of mung bean nuclease (BRL) under conditions previously reported by Zhu and Roth ( ).

    Techniques: Amplification, Purification, Labeling, DNA Sequencing, Sequencing

    Validation of Kir isoform typing with reporter enzyme ScrF I. Degenerate PCR with sub-family centred primers was performed on Kir subclone templates and products were T4 DNA polymerase end-labelled according to Materials and Methods . Diagnostic restriction digests were performed using ScrF I and an isoform specific duplex banding pattern generated consistent with the predicted banding patterns described in Figure 1 . Faint low MW fragments (

    Journal: BMC Genomics

    Article Title: Multigene family isoform profiling from blood cell lineages

    doi: 10.1186/1471-2164-3-22

    Figure Lengend Snippet: Validation of Kir isoform typing with reporter enzyme ScrF I. Degenerate PCR with sub-family centred primers was performed on Kir subclone templates and products were T4 DNA polymerase end-labelled according to Materials and Methods . Diagnostic restriction digests were performed using ScrF I and an isoform specific duplex banding pattern generated consistent with the predicted banding patterns described in Figure 1 . Faint low MW fragments (

    Article Snippet: T4 DNA Polymerase end-labelling of degenerate PCR products The products from the degenerate, sub-family centred PCR were excised and purified from agarose gels using a kit (Hybaid Ltd, Middlesex, UK) according to manufacturers protocol, and eluted into 20 μl elution solution.

    Techniques: Polymerase Chain Reaction, Diagnostic Assay, Generated

    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.

    Journal: PLoS ONE

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection

    doi: 10.1371/journal.pone.0037617

    Figure Lengend Snippet: Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.

    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.

    Techniques: Incubation, Plasmid Preparation, Clone Assay, Expressing, Construct

    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

    Preparation and analysis on circular RNA in vitro . (A) Schematic of in vitro circularization constructs. Transcripts to be circularized consist of a terminal 10 nt open loop structure (black) and a reverse-complementary repeat sequence of 11 nt, which forms an intramolecular stem (red). This structure is followed by a 63 nt constant region for detection by northern blot or PCR (blue), followed by the miRNA-122 sponge (bulge; perfect) or a scrambled control sequence (shuffle) in grey. (B) Schematic of the in vitro ligation reaction. 4-fold excess of GMP over GTP results in ∼80% of the transcripts containing a 5′-monophosphate, enabling efficient in vitro ligation by T4 RNA ligase. Ligation products are circular RNAs (intramolecular ligation) or linear dimers (intermolecular ligation). (C) In vitro ligation reactions described in (B) were analyzed on 5%, 6% or 7% polyacrylamide-urea gels by ethidium bromide staining. While mobility of linear RNAs remains unchanged compared to RNA marker, the apparent mobility of circular RNA is lower in higher percentage gels (indicated by dash/double dash or circle). (D) Purified linear or circular RNAs from (C) were transfected in HuH-7.5 cells and total RNA was prepared after 4, 8, 14, 24 and 32 h. RNAs were detected by ³²P-northern blot analysis using identical probes in the constant region [labeled blue in (A)]. (E) HuH-7.5 cells transfected with circular RNA or linear RNA from (C) were subjected to sub-cellular fractionation and cytoplasmic or nuclear fractions were analyzed by ³²P-northern blot detecting transfected RNAs along with U1 snRNA and by western blot against hnRNP A1 or GAPDH proteins as a fractionation control. In the circRNA-transfected samples, a degradation product is detected at linear monomer size (“linearized”).

    Journal: RNA Biology

    Article Title: Functional sequestration of microRNA-122 from Hepatitis C Virus by circular RNA sponges

    doi: 10.1080/15476286.2018.1435248

    Figure Lengend Snippet: Preparation and analysis on circular RNA in vitro . (A) Schematic of in vitro circularization constructs. Transcripts to be circularized consist of a terminal 10 nt open loop structure (black) and a reverse-complementary repeat sequence of 11 nt, which forms an intramolecular stem (red). This structure is followed by a 63 nt constant region for detection by northern blot or PCR (blue), followed by the miRNA-122 sponge (bulge; perfect) or a scrambled control sequence (shuffle) in grey. (B) Schematic of the in vitro ligation reaction. 4-fold excess of GMP over GTP results in ∼80% of the transcripts containing a 5′-monophosphate, enabling efficient in vitro ligation by T4 RNA ligase. Ligation products are circular RNAs (intramolecular ligation) or linear dimers (intermolecular ligation). (C) In vitro ligation reactions described in (B) were analyzed on 5%, 6% or 7% polyacrylamide-urea gels by ethidium bromide staining. While mobility of linear RNAs remains unchanged compared to RNA marker, the apparent mobility of circular RNA is lower in higher percentage gels (indicated by dash/double dash or circle). (D) Purified linear or circular RNAs from (C) were transfected in HuH-7.5 cells and total RNA was prepared after 4, 8, 14, 24 and 32 h. RNAs were detected by ³²P-northern blot analysis using identical probes in the constant region [labeled blue in (A)]. (E) HuH-7.5 cells transfected with circular RNA or linear RNA from (C) were subjected to sub-cellular fractionation and cytoplasmic or nuclear fractions were analyzed by ³²P-northern blot detecting transfected RNAs along with U1 snRNA and by western blot against hnRNP A1 or GAPDH proteins as a fractionation control. In the circRNA-transfected samples, a degradation product is detected at linear monomer size (“linearized”).

    Article Snippet: Next, T4 RNA ligase buffer and RNaseOUT (Thermo Fisher Scientific) were added and incubated for 10 min at 37°C.

    Techniques: In Vitro, Construct, Sequencing, Northern Blot, Polymerase Chain Reaction, Ligation, Staining, Marker, Purification, Transfection, Labeling, Cell Fractionation, Western Blot, Fractionation

    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.

    Journal: International Journal of Molecular Medicine

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

    doi: 10.3892/ijmm.2019.4355

    Figure Lengend 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.

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

    Techniques: Plasmid Preparation, Construct

    Plasmids pBR2TTS (A) and pBRlux‐trp (B) with the relevant restriction sites. (C) Plasmid pBRlux‐trp restores the ability of the E. coli Δ trpE strain (SM335) to grow on a tryptophan‐free medium (bottom), where the E. coli Δ trpE strain (SM301) does not grow (top).

    Journal: Microbial biotechnology

    Article Title: A bacterial reporter panel for the detection and classification of antibiotic substances

    doi: 10.1111/j.1751-7915.2012.00333.x

    Figure Lengend Snippet: Plasmids pBR2TTS (A) and pBRlux‐trp (B) with the relevant restriction sites. (C) Plasmid pBRlux‐trp restores the ability of the E. coli Δ trpE strain (SM335) to grow on a tryptophan‐free medium (bottom), where the E. coli Δ trpE strain (SM301) does not grow (top).

    Article Snippet: The PCR products were cut with restriction enzyme BSSHII (New England Biolabs, USA), and ligated into the pBR2TTS vector already harbouring trpE (T4 DNA ligase, Fermentas ), which was first transformed into E. coli DH5α and then transferred to E. coli SM301.

    Techniques: Plasmid Preparation

    Workflow of PEP. Double-stranded template DNA is blunt-end repaired using T4 DNA polymerase and T4 polynucleotide kinase (not depicted). ( I ) Using T4 DNA ligase, biotinylated adapters are attached to both ends of the template molecules. The blunt end ligation reaction also produces adapter dimers, which are subsequently removed by size selective purification. ( II ) 5′-tailed primers carrying the 454 ‘B’ sequence (shown in blue) are hybridized to the overhanging 3′-ends of the adapters. Primer extension is carried out under reaction conditions optimal for the assayed polymerase. Unless second-strand synthesis stops prematurely, due to a blocking lesion, a nick or random polymerase stalling, the flanking adapter sequence (shown in red) is copied. ( III ) Primer extension products are captured on streptavidine beads to remove excess primers and extension products from nicked template strands. Extension products are released by heat denaturation. ( IV ) A 454 sequencing library is created by attaching single-stranded adapters with the 454 ‘A’ sequence (shown in green) to the 3′-ends. The sequencing library is converted to double-stranded form (not depicted) to allow for efficient removal of excess A-adapters. The 454 sequencing is initiated from the A-adapter. If primer extensions were complete, sequences will start with an 8-bp adapter sequence, which serves as the end-of-template recognition sequence (framed by rectangles).

    Journal: Nucleic Acids Research

    Article Title: Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA

    doi: 10.1093/nar/gkq572

    Figure Lengend Snippet: Workflow of PEP. Double-stranded template DNA is blunt-end repaired using T4 DNA polymerase and T4 polynucleotide kinase (not depicted). ( I ) Using T4 DNA ligase, biotinylated adapters are attached to both ends of the template molecules. The blunt end ligation reaction also produces adapter dimers, which are subsequently removed by size selective purification. ( II ) 5′-tailed primers carrying the 454 ‘B’ sequence (shown in blue) are hybridized to the overhanging 3′-ends of the adapters. Primer extension is carried out under reaction conditions optimal for the assayed polymerase. Unless second-strand synthesis stops prematurely, due to a blocking lesion, a nick or random polymerase stalling, the flanking adapter sequence (shown in red) is copied. ( III ) Primer extension products are captured on streptavidine beads to remove excess primers and extension products from nicked template strands. Extension products are released by heat denaturation. ( IV ) A 454 sequencing library is created by attaching single-stranded adapters with the 454 ‘A’ sequence (shown in green) to the 3′-ends. The sequencing library is converted to double-stranded form (not depicted) to allow for efficient removal of excess A-adapters. The 454 sequencing is initiated from the A-adapter. If primer extensions were complete, sequences will start with an 8-bp adapter sequence, which serves as the end-of-template recognition sequence (framed by rectangles).

    Article Snippet: PEP assays and sequencing For blunt end repair, ∼15 ng of PCR product pool, 2 ng of fragmented horse DNA, 4 ng of UV-irradiated horse DNA, 10 µl ancient DNA extract or a water sample were incubated for 15 min at 12°C and 15 min at 25°C in a 40 µl reaction containing in final concentrations 1× Tango buffer, 0.1 U/µl T4 DNA polymerase, 0.5 U/µl T4 polynucleotide kinase (all Fermentas), 1 mM ATP and 0.1 mM dNTP.

    Techniques: Ligation, Purification, Sequencing, Blocking Assay

    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