t4 dna ligase buffer  (New England Biolabs)


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    T4 DNA Ligase
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    T4 DNA Ligase 100 000 units
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    New England Biolabs t4 dna ligase buffer
    T4 DNA Ligase
    T4 DNA Ligase 100 000 units
    https://www.bioz.com/result/t4 dna ligase buffer/product/New England Biolabs
    Average 99 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase buffer - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes"

    Article Title: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes

    Journal: Plant Methods

    doi: 10.1186/s13007-018-0359-7

    Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts
    Figure Legend Snippet: Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts

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

    2) Product Images from "Demonstration of a universal surface DNA computer"

    Article Title: Demonstration of a universal surface DNA computer

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh635

    Computation of a simple circuit consisting of a NOR gate and an OR gate. ( a ) DNAs encoding the inputs were attached to the surfaces by the 3′ thiol group. ‘FFX’ designates the input DNAs in which X 1 is FALSE, X 2 is FALSE and X3 can be either TRUE or FALSE (i.e. input A and B). P indicates a phosphate group and S the underlying surface chemistry. ( b ) Complements to the words (X 1 is F) and (X 2 is F) were added to the surfaces and allowed to hybridize with the attached DNAs. ( c ) T4 DNA ligase linked the two complements hybridized with the ‘FFX’ DNAs. ( d ) Differential melting melted off hybridized one-word complements and left the linked two-word complements in place. ( e ) In the ‘FFX’ DNAs, polymerase extended the two-word complements to their 3′ ends. A TRUE fourth word (X 4 is T) was appended to the distal end of the now double-stranded ‘FFX’ DNAs. Note here that the 5′ end of the newly attached word was not phosphorylated, whereas the 5′ end of its complement was phosphorylated. This was designed so that only one copy of this word would be appended. ( f ) The complements to the words (X 1 is T) and (X 2 is T) were added to the surface and allowed to hybridize. ( g ) The complements added in (f) were extended by polymerase. Then a FALSE fourth word was appended to the surfaces by ligation. Note again that the design of this one-bit word was such that only the 5′ end of its complementary strand was phosphorylated for the same reason as for (e). The lack of a 5′ phosphate at the ends of the ‘FFX’ DNAs also ensured that this FALSE fourth word would not be appended to them. ( h ) DNAs after the NOR gate computation. Note here that the DNAs were now without a phosphate group. A kinase phosphorylated them before the OR gate computation. ( i ) DNAs in which (X 3 is F) and (X 4 is F) were MARKED in the first APPEND-MARKED operation for the OR gate. ( j ) A FALSE fifth word, rather than a TRUE fifth word, was appended. The change is equivalent to a NOT gate. ( k ) DNAs after the OR gate computation.
    Figure Legend Snippet: Computation of a simple circuit consisting of a NOR gate and an OR gate. ( a ) DNAs encoding the inputs were attached to the surfaces by the 3′ thiol group. ‘FFX’ designates the input DNAs in which X 1 is FALSE, X 2 is FALSE and X3 can be either TRUE or FALSE (i.e. input A and B). P indicates a phosphate group and S the underlying surface chemistry. ( b ) Complements to the words (X 1 is F) and (X 2 is F) were added to the surfaces and allowed to hybridize with the attached DNAs. ( c ) T4 DNA ligase linked the two complements hybridized with the ‘FFX’ DNAs. ( d ) Differential melting melted off hybridized one-word complements and left the linked two-word complements in place. ( e ) In the ‘FFX’ DNAs, polymerase extended the two-word complements to their 3′ ends. A TRUE fourth word (X 4 is T) was appended to the distal end of the now double-stranded ‘FFX’ DNAs. Note here that the 5′ end of the newly attached word was not phosphorylated, whereas the 5′ end of its complement was phosphorylated. This was designed so that only one copy of this word would be appended. ( f ) The complements to the words (X 1 is T) and (X 2 is T) were added to the surface and allowed to hybridize. ( g ) The complements added in (f) were extended by polymerase. Then a FALSE fourth word was appended to the surfaces by ligation. Note again that the design of this one-bit word was such that only the 5′ end of its complementary strand was phosphorylated for the same reason as for (e). The lack of a 5′ phosphate at the ends of the ‘FFX’ DNAs also ensured that this FALSE fourth word would not be appended to them. ( h ) DNAs after the NOR gate computation. Note here that the DNAs were now without a phosphate group. A kinase phosphorylated them before the OR gate computation. ( i ) DNAs in which (X 3 is F) and (X 4 is F) were MARKED in the first APPEND-MARKED operation for the OR gate. ( j ) A FALSE fifth word, rather than a TRUE fifth word, was appended. The change is equivalent to a NOT gate. ( k ) DNAs after the OR gate computation.

    Techniques Used: Ligation

    3) Product Images from "Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes"

    Article Title: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes

    Journal: Plant Methods

    doi: 10.1186/s13007-018-0359-7

    Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts
    Figure Legend Snippet: Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts

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

    4) Product Images from "Analysis of polyclonal vector integration sites using Nanopore sequencing as a scalable, cost-effective platform"

    Article Title: Analysis of polyclonal vector integration sites using Nanopore sequencing as a scalable, cost-effective platform

    Journal: bioRxiv

    doi: 10.1101/833897

    Schematic for PCR amplification of flanking genomic sequences. (A) Genomic DNA is digested with two 6-cutter restriction enzymes, NcoI and BspHI , which together are anticipated to cut at approximately 2 kb intervals. There are 4 restriction sites within the transgene sequence, the most distal of which is 1185 bp from the 3’LTR / genomic junction. NcoI and BspHI generate identical 4-nucleotide 5’ overhangs: 5’-CATG-3’, which can be circularized for inverse PCR or ligated to linker cassettes. (B) Inverse PCR begins with circularization with T4 DNA ligase, followed by PCR amplification of the unknown flanking genomic sequences using primers targeting the 3’LTR and the 3’LTR/distal transgene junction. This is followed by nested PCR, which incorporates tailing sequences for subsequent barcoding. The combined lengths of the dotted lines in the inner circle indicate the minimum theoretical length prior to the addition of tailing sequences and barcodes. (C) The ligation cassette comprises two partially complementary strands: a 27-nucleotide strand and a 14-nucleotide strand, the latter with a mismatched A at the 3’ end and a 5’overhang (5’-ATG-3’). Before cassette ligation, the genomic DNA fragments are filled with a single ddCTP to prevent elongation or ligation at the recessed 3’ end. Cassette ligation results in a nick on this strand, indicated by ‘X’. During the first cycle of PCR, fragments containing flanking genomic DNA are amplified by a primer spanning the transgene/3’LTR. The longer cassette strand does not prime because its complementary shorter strand has not ligated; whereas the shorter cassette strand does not prime because only 10 nucleotides are complementary to the longer cassette strand, resulting in a high annealing temperature. This cassette design limits the amplification of non-flanking genomic DNA and reduces PCR blocking by the shorter cassette strand. Subsequent cycles are primed by both the transgene/3’LTR primer and the longer cassette strand.
    Figure Legend Snippet: Schematic for PCR amplification of flanking genomic sequences. (A) Genomic DNA is digested with two 6-cutter restriction enzymes, NcoI and BspHI , which together are anticipated to cut at approximately 2 kb intervals. There are 4 restriction sites within the transgene sequence, the most distal of which is 1185 bp from the 3’LTR / genomic junction. NcoI and BspHI generate identical 4-nucleotide 5’ overhangs: 5’-CATG-3’, which can be circularized for inverse PCR or ligated to linker cassettes. (B) Inverse PCR begins with circularization with T4 DNA ligase, followed by PCR amplification of the unknown flanking genomic sequences using primers targeting the 3’LTR and the 3’LTR/distal transgene junction. This is followed by nested PCR, which incorporates tailing sequences for subsequent barcoding. The combined lengths of the dotted lines in the inner circle indicate the minimum theoretical length prior to the addition of tailing sequences and barcodes. (C) The ligation cassette comprises two partially complementary strands: a 27-nucleotide strand and a 14-nucleotide strand, the latter with a mismatched A at the 3’ end and a 5’overhang (5’-ATG-3’). Before cassette ligation, the genomic DNA fragments are filled with a single ddCTP to prevent elongation or ligation at the recessed 3’ end. Cassette ligation results in a nick on this strand, indicated by ‘X’. During the first cycle of PCR, fragments containing flanking genomic DNA are amplified by a primer spanning the transgene/3’LTR. The longer cassette strand does not prime because its complementary shorter strand has not ligated; whereas the shorter cassette strand does not prime because only 10 nucleotides are complementary to the longer cassette strand, resulting in a high annealing temperature. This cassette design limits the amplification of non-flanking genomic DNA and reduces PCR blocking by the shorter cassette strand. Subsequent cycles are primed by both the transgene/3’LTR primer and the longer cassette strand.

    Techniques Used: Polymerase Chain Reaction, Amplification, Genomic Sequencing, Sequencing, Inverse PCR, Nested PCR, Ligation, Blocking Assay

    5) Product Images from "Structure-seq2: sensitive and accurate genome-wide profiling of RNA structure in vivo"

    Article Title: Structure-seq2: sensitive and accurate genome-wide profiling of RNA structure in vivo

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx533

    Structure-seq2 leads to a lower ligation bias. ( A ) After RT (Figure 1 , step 1A/1B), excess of the 27 nt primer (blue, top, right) is still present in the solution. During ligation (Figure 1 , step 3A/3B), this primer can also ligate to the 40 nt hairpin adaptor (pink) to form an unwanted 67 nt by-product which has no insert and so results in sequencing reads with no utility. ( B ) The complement of the first nucleotide after the adaptor sequence read during sequencing is the nucleotide that ligated to the adaptor. Our new T4 DNA ligase-based method (green, –DMS and pink, +DMS) substantially decreases ligation bias as compared to the previous Circligase-based method (blue). Percentages equaling the transcriptomic distribution of the four nucleotides (black) are ideal.
    Figure Legend Snippet: Structure-seq2 leads to a lower ligation bias. ( A ) After RT (Figure 1 , step 1A/1B), excess of the 27 nt primer (blue, top, right) is still present in the solution. During ligation (Figure 1 , step 3A/3B), this primer can also ligate to the 40 nt hairpin adaptor (pink) to form an unwanted 67 nt by-product which has no insert and so results in sequencing reads with no utility. ( B ) The complement of the first nucleotide after the adaptor sequence read during sequencing is the nucleotide that ligated to the adaptor. Our new T4 DNA ligase-based method (green, –DMS and pink, +DMS) substantially decreases ligation bias as compared to the previous Circligase-based method (blue). Percentages equaling the transcriptomic distribution of the four nucleotides (black) are ideal.

    Techniques Used: Ligation, Sequencing

    Two versions of Structure-seq2 produce high quality data. In Structure-seq2, RNA (kelly green) is first modified by DMS or another chemical that can be read-out through reverse transcription. The RNA is then prepared for Illumina NGS sequencing by conversion to cDNA (Step 1A/1B, blue), ligating an adaptor (Step 3A/3B), and amplifying the products while incorporating TruSeq primer sequences (Step 5A/5B). In order to increase library quality, numerous improvements were made to the original Structure-seq protocol (boxed). These include performing the ligation with a hairpin adaptor and T4 DNA ligase (Step 3A/3B; pink) ( 10 ), and adding various purification steps to remove a deleterious by-product (Figure 2A ). We present two options for purification: PAGE purification ( A ) or a biotin–streptavidin pull down ( B ). In the PAGE purification method, an additional PAGE purification step is added after reverse transcription (Step 2A). In the biotin–streptavidin pull down method, biotinylated dNTPs (cyan) are incorporated into the extended product during reverse transcription (Step 1B) and are purified via a magnetic streptavidin pull down after reverse transcription (Step 2B) and after ligation (Step 4B). There is also a common, final PAGE purification step following amplification (Step 5A/5B). Finally, a custom sequencing primer (light green) is used during sequencing (Step 7A/7B) to further provide high quality data. Supplementary Figure S1 is a version of this figure with all the nucleotides shown explicitly.
    Figure Legend Snippet: Two versions of Structure-seq2 produce high quality data. In Structure-seq2, RNA (kelly green) is first modified by DMS or another chemical that can be read-out through reverse transcription. The RNA is then prepared for Illumina NGS sequencing by conversion to cDNA (Step 1A/1B, blue), ligating an adaptor (Step 3A/3B), and amplifying the products while incorporating TruSeq primer sequences (Step 5A/5B). In order to increase library quality, numerous improvements were made to the original Structure-seq protocol (boxed). These include performing the ligation with a hairpin adaptor and T4 DNA ligase (Step 3A/3B; pink) ( 10 ), and adding various purification steps to remove a deleterious by-product (Figure 2A ). We present two options for purification: PAGE purification ( A ) or a biotin–streptavidin pull down ( B ). In the PAGE purification method, an additional PAGE purification step is added after reverse transcription (Step 2A). In the biotin–streptavidin pull down method, biotinylated dNTPs (cyan) are incorporated into the extended product during reverse transcription (Step 1B) and are purified via a magnetic streptavidin pull down after reverse transcription (Step 2B) and after ligation (Step 4B). There is also a common, final PAGE purification step following amplification (Step 5A/5B). Finally, a custom sequencing primer (light green) is used during sequencing (Step 7A/7B) to further provide high quality data. Supplementary Figure S1 is a version of this figure with all the nucleotides shown explicitly.

    Techniques Used: Modification, Next-Generation Sequencing, Sequencing, Ligation, Purification, Polyacrylamide Gel Electrophoresis, Amplification

    6) Product Images from "Structure-seq2: sensitive and accurate genome-wide profiling of RNA structure in vivo"

    Article Title: Structure-seq2: sensitive and accurate genome-wide profiling of RNA structure in vivo

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx533

    Structure-seq2 leads to a lower ligation bias. ( A ) After RT (Figure 1 , step 1A/1B), excess of the 27 nt primer (blue, top, right) is still present in the solution. During ligation (Figure 1 , step 3A/3B), this primer can also ligate to the 40 nt hairpin adaptor (pink) to form an unwanted 67 nt by-product which has no insert and so results in sequencing reads with no utility. ( B ) The complement of the first nucleotide after the adaptor sequence read during sequencing is the nucleotide that ligated to the adaptor. Our new T4 DNA ligase-based method (green, –DMS and pink, +DMS) substantially decreases ligation bias as compared to the previous Circligase-based method (blue). Percentages equaling the transcriptomic distribution of the four nucleotides (black) are ideal.
    Figure Legend Snippet: Structure-seq2 leads to a lower ligation bias. ( A ) After RT (Figure 1 , step 1A/1B), excess of the 27 nt primer (blue, top, right) is still present in the solution. During ligation (Figure 1 , step 3A/3B), this primer can also ligate to the 40 nt hairpin adaptor (pink) to form an unwanted 67 nt by-product which has no insert and so results in sequencing reads with no utility. ( B ) The complement of the first nucleotide after the adaptor sequence read during sequencing is the nucleotide that ligated to the adaptor. Our new T4 DNA ligase-based method (green, –DMS and pink, +DMS) substantially decreases ligation bias as compared to the previous Circligase-based method (blue). Percentages equaling the transcriptomic distribution of the four nucleotides (black) are ideal.

    Techniques Used: Ligation, Sequencing

    Two versions of Structure-seq2 produce high quality data. In Structure-seq2, RNA (kelly green) is first modified by DMS or another chemical that can be read-out through reverse transcription. The RNA is then prepared for Illumina NGS sequencing by conversion to cDNA (Step 1A/1B, blue), ligating an adaptor (Step 3A/3B), and amplifying the products while incorporating TruSeq primer sequences (Step 5A/5B). In order to increase library quality, numerous improvements were made to the original Structure-seq protocol (boxed). These include performing the ligation with a hairpin adaptor and T4 DNA ligase (Step 3A/3B; pink) ( 10 ), and adding various purification steps to remove a deleterious by-product (Figure 2A ). We present two options for purification: PAGE purification ( A ) or a biotin–streptavidin pull down ( B ). In the PAGE purification method, an additional PAGE purification step is added after reverse transcription (Step 2A). In the biotin–streptavidin pull down method, biotinylated dNTPs (cyan) are incorporated into the extended product during reverse transcription (Step 1B) and are purified via a magnetic streptavidin pull down after reverse transcription (Step 2B) and after ligation (Step 4B). There is also a common, final PAGE purification step following amplification (Step 5A/5B). Finally, a custom sequencing primer (light green) is used during sequencing (Step 7A/7B) to further provide high quality data. Supplementary Figure S1 is a version of this figure with all the nucleotides shown explicitly.
    Figure Legend Snippet: Two versions of Structure-seq2 produce high quality data. In Structure-seq2, RNA (kelly green) is first modified by DMS or another chemical that can be read-out through reverse transcription. The RNA is then prepared for Illumina NGS sequencing by conversion to cDNA (Step 1A/1B, blue), ligating an adaptor (Step 3A/3B), and amplifying the products while incorporating TruSeq primer sequences (Step 5A/5B). In order to increase library quality, numerous improvements were made to the original Structure-seq protocol (boxed). These include performing the ligation with a hairpin adaptor and T4 DNA ligase (Step 3A/3B; pink) ( 10 ), and adding various purification steps to remove a deleterious by-product (Figure 2A ). We present two options for purification: PAGE purification ( A ) or a biotin–streptavidin pull down ( B ). In the PAGE purification method, an additional PAGE purification step is added after reverse transcription (Step 2A). In the biotin–streptavidin pull down method, biotinylated dNTPs (cyan) are incorporated into the extended product during reverse transcription (Step 1B) and are purified via a magnetic streptavidin pull down after reverse transcription (Step 2B) and after ligation (Step 4B). There is also a common, final PAGE purification step following amplification (Step 5A/5B). Finally, a custom sequencing primer (light green) is used during sequencing (Step 7A/7B) to further provide high quality data. Supplementary Figure S1 is a version of this figure with all the nucleotides shown explicitly.

    Techniques Used: Modification, Next-Generation Sequencing, Sequencing, Ligation, Purification, Polyacrylamide Gel Electrophoresis, Amplification

    7) Product Images from "Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes"

    Article Title: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes

    Journal: Plant Methods

    doi: 10.1186/s13007-018-0359-7

    Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts
    Figure Legend Snippet: Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts

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

    8) Product Images from "Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes"

    Article Title: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes

    Journal: Plant Methods

    doi: 10.1186/s13007-018-0359-7

    Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts
    Figure Legend Snippet: Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts

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

    9) Product Images from "Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes"

    Article Title: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes

    Journal: Plant Methods

    doi: 10.1186/s13007-018-0359-7

    Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts
    Figure Legend Snippet: Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts

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

    10) Product Images from "Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes"

    Article Title: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes

    Journal: Plant Methods

    doi: 10.1186/s13007-018-0359-7

    Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts
    Figure Legend Snippet: Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts

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

    11) Product Images from "Enhancement of DNA flexibility in vitro and in vivo by HMGB box A proteins carrying box B residues"

    Article Title: Enhancement of DNA flexibility in vitro and in vivo by HMGB box A proteins carrying box B residues

    Journal: Biochemistry

    doi: 10.1021/bi802269f

    In vitro assay of DNA flexibility enhancement by HMGB proteins and chimeras. A. Example data from T4 DNA ligase cyclization assay for 200-bp DNA probe in the absence (—) and presence of 40 nM HMGB constructs 16 and 5 (see ). B. Graphical
    Figure Legend Snippet: In vitro assay of DNA flexibility enhancement by HMGB proteins and chimeras. A. Example data from T4 DNA ligase cyclization assay for 200-bp DNA probe in the absence (—) and presence of 40 nM HMGB constructs 16 and 5 (see ). B. Graphical

    Techniques Used: In Vitro, Construct

    12) Product Images from "Demonstration of a universal surface DNA computer"

    Article Title: Demonstration of a universal surface DNA computer

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh635

    Computation of a simple circuit consisting of a NOR gate and an OR gate. ( a ) DNAs encoding the inputs were attached to the surfaces by the 3′ thiol group. ‘FFX’ designates the input DNAs in which X 1 is FALSE, X 2 is FALSE and X3 can be either TRUE or FALSE (i.e. input A and B). P indicates a phosphate group and S the underlying surface chemistry. ( b ) Complements to the words (X 1 is F) and (X 2 is F) were added to the surfaces and allowed to hybridize with the attached DNAs. ( c ) T4 DNA ligase linked the two complements hybridized with the ‘FFX’ DNAs. ( d ) Differential melting melted off hybridized one-word complements and left the linked two-word complements in place. ( e ) In the ‘FFX’ DNAs, polymerase extended the two-word complements to their 3′ ends. A TRUE fourth word (X 4 is T) was appended to the distal end of the now double-stranded ‘FFX’ DNAs. Note here that the 5′ end of the newly attached word was not phosphorylated, whereas the 5′ end of its complement was phosphorylated. This was designed so that only one copy of this word would be appended. ( f ) The complements to the words (X 1 is T) and (X 2 is T) were added to the surface and allowed to hybridize. ( g ) The complements added in (f) were extended by polymerase. Then a FALSE fourth word was appended to the surfaces by ligation. Note again that the design of this one-bit word was such that only the 5′ end of its complementary strand was phosphorylated for the same reason as for (e). The lack of a 5′ phosphate at the ends of the ‘FFX’ DNAs also ensured that this FALSE fourth word would not be appended to them. ( h ) DNAs after the NOR gate computation. Note here that the DNAs were now without a phosphate group. A kinase phosphorylated them before the OR gate computation. ( i ) DNAs in which (X 3 is F) and (X 4 is F) were MARKED in the first APPEND-MARKED operation for the OR gate. ( j ) A FALSE fifth word, rather than a TRUE fifth word, was appended. The change is equivalent to a NOT gate. ( k ) DNAs after the OR gate computation.
    Figure Legend Snippet: Computation of a simple circuit consisting of a NOR gate and an OR gate. ( a ) DNAs encoding the inputs were attached to the surfaces by the 3′ thiol group. ‘FFX’ designates the input DNAs in which X 1 is FALSE, X 2 is FALSE and X3 can be either TRUE or FALSE (i.e. input A and B). P indicates a phosphate group and S the underlying surface chemistry. ( b ) Complements to the words (X 1 is F) and (X 2 is F) were added to the surfaces and allowed to hybridize with the attached DNAs. ( c ) T4 DNA ligase linked the two complements hybridized with the ‘FFX’ DNAs. ( d ) Differential melting melted off hybridized one-word complements and left the linked two-word complements in place. ( e ) In the ‘FFX’ DNAs, polymerase extended the two-word complements to their 3′ ends. A TRUE fourth word (X 4 is T) was appended to the distal end of the now double-stranded ‘FFX’ DNAs. Note here that the 5′ end of the newly attached word was not phosphorylated, whereas the 5′ end of its complement was phosphorylated. This was designed so that only one copy of this word would be appended. ( f ) The complements to the words (X 1 is T) and (X 2 is T) were added to the surface and allowed to hybridize. ( g ) The complements added in (f) were extended by polymerase. Then a FALSE fourth word was appended to the surfaces by ligation. Note again that the design of this one-bit word was such that only the 5′ end of its complementary strand was phosphorylated for the same reason as for (e). The lack of a 5′ phosphate at the ends of the ‘FFX’ DNAs also ensured that this FALSE fourth word would not be appended to them. ( h ) DNAs after the NOR gate computation. Note here that the DNAs were now without a phosphate group. A kinase phosphorylated them before the OR gate computation. ( i ) DNAs in which (X 3 is F) and (X 4 is F) were MARKED in the first APPEND-MARKED operation for the OR gate. ( j ) A FALSE fifth word, rather than a TRUE fifth word, was appended. The change is equivalent to a NOT gate. ( k ) DNAs after the OR gate computation.

    Techniques Used: Ligation

    13) Product Images from "Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes"

    Article Title: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes

    Journal: Plant Methods

    doi: 10.1186/s13007-018-0359-7

    Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts
    Figure Legend Snippet: Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts

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

    14) Product Images from "Nucleic acid evolution and minimization by nonhomologous random recombination"

    Article Title: Nucleic acid evolution and minimization by nonhomologous random recombination

    Journal: Nature biotechnology

    doi: 10.1038/nbt736

    Overview of the nonhomologous random recombination (NRR) method. (A) Starting DNA sequences are randomly digested with DNase I, blunt-ended with T4 DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.
    Figure Legend Snippet: Overview of the nonhomologous random recombination (NRR) method. (A) Starting DNA sequences are randomly digested with DNase I, blunt-ended with T4 DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.

    Techniques Used: Ligation, Binding Assay

    15) Product Images from "S1-seq assay for mapping processed DNA ends"

    Article Title: S1-seq assay for mapping processed DNA ends

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2017.11.031

    Schematic of the key steps employed in S1-seq: DNA from meiotic cells bearing processed Spo11 DSBs is embedded in agarose plugs to protect from shearing. Treatment with S1 nuclease and T4 DNA polymerase removes the 3′ overhang and prepares the ends for ligation with the 5′ biotinylated adaptor. Following extraction from the plugs and sonication, the biotinylated fragments are bound on streptavidin beads and the second adaptor is ligated. Low-cycle amplification by PCR creates the library that is submitted for next generation sequencing and eventually mapped to the reference genome.
    Figure Legend Snippet: Schematic of the key steps employed in S1-seq: DNA from meiotic cells bearing processed Spo11 DSBs is embedded in agarose plugs to protect from shearing. Treatment with S1 nuclease and T4 DNA polymerase removes the 3′ overhang and prepares the ends for ligation with the 5′ biotinylated adaptor. Following extraction from the plugs and sonication, the biotinylated fragments are bound on streptavidin beads and the second adaptor is ligated. Low-cycle amplification by PCR creates the library that is submitted for next generation sequencing and eventually mapped to the reference genome.

    Techniques Used: Ligation, Sonication, Amplification, Polymerase Chain Reaction, Next-Generation Sequencing

    16) Product Images from "Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes"

    Article Title: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes

    Journal: Plant Methods

    doi: 10.1186/s13007-018-0359-7

    Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts
    Figure Legend Snippet: Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts

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

    17) Product Images from "Efficient assembly of very short oligonucleotides using T4 DNA Ligase"

    Article Title: Efficient assembly of very short oligonucleotides using T4 DNA Ligase

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-3-291

    Enhancement of T4 DNA ligase activity by supplemental oligonucleotides. (a) Unsuccessful 4-bp duplex reactions could be salvaged by utilizing a supplementary oligonucleotide, designed to complement the first oligonucleotide-dsDNA duplex but is unphosphorylated to prevent ligation of itself. Two hour ligation of the 4-bp reaction at 16°C supplemented with 3.33 μM of the hexamer, shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (b) Ligation reaction of an octamer supplemented with a second octamer in which one is used for ligation and the other is used to extend the duplex. A two hour ligation at 16°C of serial concentrations of the octamer with 3.33 μM of the supplementary octamer shows significant ligation (■) compared to reactions without the supplemental octamer (◆). (c) Unsuccessful 3-bp duplex reactions could be salvaged by utilizing a supplementary hexamer that hybridized at all six positions. A two hour ligation of the 3-bp reaction at 16°C with 3.33 μM supplementary hexamer shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (d) Ligation using a hexamer pair at 4°C for 16 hours shows limited improvement (■) compared to the unsupplemented (◆) control.
    Figure Legend Snippet: Enhancement of T4 DNA ligase activity by supplemental oligonucleotides. (a) Unsuccessful 4-bp duplex reactions could be salvaged by utilizing a supplementary oligonucleotide, designed to complement the first oligonucleotide-dsDNA duplex but is unphosphorylated to prevent ligation of itself. Two hour ligation of the 4-bp reaction at 16°C supplemented with 3.33 μM of the hexamer, shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (b) Ligation reaction of an octamer supplemented with a second octamer in which one is used for ligation and the other is used to extend the duplex. A two hour ligation at 16°C of serial concentrations of the octamer with 3.33 μM of the supplementary octamer shows significant ligation (■) compared to reactions without the supplemental octamer (◆). (c) Unsuccessful 3-bp duplex reactions could be salvaged by utilizing a supplementary hexamer that hybridized at all six positions. A two hour ligation of the 3-bp reaction at 16°C with 3.33 μM supplementary hexamer shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (d) Ligation using a hexamer pair at 4°C for 16 hours shows limited improvement (■) compared to the unsupplemented (◆) control.

    Techniques Used: Activity Assay, Ligation

    Evaluation of minimal oligonucleotide substrate requirements for T4 DNA ligase. (a) Schematic diagram of an immobilized DNA strand used in ligation assays and DNA construction. M-270 Dynabeads (Invitrogen) are attached through a streptavidin-biotin linkage to the 5' end of a double stranded DNA. The free end is designed with a variable 5' overhang, complementary to labeled oligonucleotides used in ligation. An additional BbsI restriction site and a forward primer site are included in the case of DNA construction. (b) Increasing concentrations of 5'-phosphorylated, 3'-fluorescently labeled oligonucleotide are ligated to 5 pmoles of immobilized dsDNA with a complementary overhang. Reactions were performed for one hour at 16°C and washed with TE to remove unligated substrate. Successful ligation kinetics are observed at the 5-bp duplex length (▲), but no significant ligation occurs at lengths of 4-bp (■) or 3-bp (◆).
    Figure Legend Snippet: Evaluation of minimal oligonucleotide substrate requirements for T4 DNA ligase. (a) Schematic diagram of an immobilized DNA strand used in ligation assays and DNA construction. M-270 Dynabeads (Invitrogen) are attached through a streptavidin-biotin linkage to the 5' end of a double stranded DNA. The free end is designed with a variable 5' overhang, complementary to labeled oligonucleotides used in ligation. An additional BbsI restriction site and a forward primer site are included in the case of DNA construction. (b) Increasing concentrations of 5'-phosphorylated, 3'-fluorescently labeled oligonucleotide are ligated to 5 pmoles of immobilized dsDNA with a complementary overhang. Reactions were performed for one hour at 16°C and washed with TE to remove unligated substrate. Successful ligation kinetics are observed at the 5-bp duplex length (▲), but no significant ligation occurs at lengths of 4-bp (■) or 3-bp (◆).

    Techniques Used: Ligation, Labeling

    Related Articles

    DNA Ligation:

    Article Title: Mechanical properties of DNA-like polymers
    Article Snippet: .. DNA ligase-catalyzed cyclization reactions were performed at ∼22°C with 1 nM DNA restriction fragment, T4 DNA ligation buffer (50 mM Tris–HCl, pH 7.5, 10 mM MgCl2 , 1 mM ATP, 10 mM dithiothreitol; NEB) and a final concentration of 100 U/ml T4 DNA ligase (NEB). .. Aliquots (10 µl) were removed at 5, 10, 15 and 20 min time points, quenched by addition of EDTA to 20 mM and then analyzed by electrophoresis through 5% native polyacrylamide gels (29:1 acrylamide:bisacylamide, BioRad) in 0.5× TBE buffer (50 mM Tris base, 55 mM boric acid and 1 mM EDTA, pH 8.3), followed by drying and storage phosphor imaging.

    Ligation:

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles
    Article Snippet: .. Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA). .. Animals and tissues Thirteen-week old male C57BL/6 J mice (n = 3) purchased from CLEA Japan Inc. (CLEA Japan Inc., Tokyo, Japan) were sacrificed by cervical dislocation to collect liver, kidney, and hippocampus samples.

    Magnetic Beads:

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles
    Article Snippet: .. Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA). .. Animals and tissues Thirteen-week old male C57BL/6 J mice (n = 3) purchased from CLEA Japan Inc. (CLEA Japan Inc., Tokyo, Japan) were sacrificed by cervical dislocation to collect liver, kidney, and hippocampus samples.

    SYBR Green Assay:

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles
    Article Snippet: .. Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA). .. Animals and tissues Thirteen-week old male C57BL/6 J mice (n = 3) purchased from CLEA Japan Inc. (CLEA Japan Inc., Tokyo, Japan) were sacrificed by cervical dislocation to collect liver, kidney, and hippocampus samples.

    Concentration Assay:

    Article Title: Mechanical properties of DNA-like polymers
    Article Snippet: .. DNA ligase-catalyzed cyclization reactions were performed at ∼22°C with 1 nM DNA restriction fragment, T4 DNA ligation buffer (50 mM Tris–HCl, pH 7.5, 10 mM MgCl2 , 1 mM ATP, 10 mM dithiothreitol; NEB) and a final concentration of 100 U/ml T4 DNA ligase (NEB). .. Aliquots (10 µl) were removed at 5, 10, 15 and 20 min time points, quenched by addition of EDTA to 20 mM and then analyzed by electrophoresis through 5% native polyacrylamide gels (29:1 acrylamide:bisacylamide, BioRad) in 0.5× TBE buffer (50 mM Tris base, 55 mM boric acid and 1 mM EDTA, pH 8.3), followed by drying and storage phosphor imaging.

    Incubation:

    Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression
    Article Snippet: .. 200 ng of this DNA was incubated with nuclear extracts for 1h at 25°C in a reaction mixture containing 1 × ligase buffer and 1 μl of T4 DNA ligase (200 U, New England Biolabs). .. Reactions were impeded and de-proteinated by adding Proteinase K enzyme (Invitrogen) followed by 15 min incubation at 37°C.

    other:

    Article Title: Synapsis of Recombination Signal Sequences Located in cis and DNA Underwinding in V(D)J Recombination
    Article Snippet: T4 DNA ligase was then added, and all subsequent steps were performed as described above.

    Methylation:

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles
    Article Snippet: .. Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA). .. Animals and tissues Thirteen-week old male C57BL/6 J mice (n = 3) purchased from CLEA Japan Inc. (CLEA Japan Inc., Tokyo, Japan) were sacrificed by cervical dislocation to collect liver, kidney, and hippocampus samples.

    Sequencing:

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles
    Article Snippet: .. Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA). .. Animals and tissues Thirteen-week old male C57BL/6 J mice (n = 3) purchased from CLEA Japan Inc. (CLEA Japan Inc., Tokyo, Japan) were sacrificed by cervical dislocation to collect liver, kidney, and hippocampus samples.

    Plasmid Preparation:

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles
    Article Snippet: .. Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA). .. Animals and tissues Thirteen-week old male C57BL/6 J mice (n = 3) purchased from CLEA Japan Inc. (CLEA Japan Inc., Tokyo, Japan) were sacrificed by cervical dislocation to collect liver, kidney, and hippocampus samples.

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    New England Biolabs t4 dna ligase reaction buffer
    Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of <t>T4</t> DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.
    T4 Dna Ligase Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase reaction buffer/product/New England Biolabs
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    93
    New England Biolabs t4 ligase buffer
    RECQ1 modulates DNA end-joining. A. RECQ1 affects DNA end-joining by T4 ligase. Comparison of ligation products of 5′-cohesive (left panel) or blunt (right panel) ended linear DNA after incubation with <t>T4</t> ligase alone, or after pre-incubation with the increasing amount of RECQ1 (0–260 ng) or Ku70/80 (0–400 ng) as described in materials and methods. IgG (1µg) was included as unrelated protein in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. O.C., open circular. DNA size marker is shown in leftmost lane. B. RECQ1 antibody interferes with cell free extract mediated end-joining in vitro . The effect of antibodies against human RECQ1 (1.5 and 3 µg) or Ku70/80 (1 and 2 µg) was tested in DNA end-joining reactions assembled with 5 µg HeLa cell free extract. Antibodies at the indicated amounts were incubated with cell free extract for 10 min at room temperature in NHEJ buffer without DNA and ATP. Subsequently, EcoRI-linearized pUC19 DNA and ATP were added to start the reaction followed by 2 h incubation at room temperature. Reaction products were purified and analyzed by SYBR Gold staining after resolution on agarose gel. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. C. Immunodepletion of RECQ1 or Ku70/80 from cell extracts results in similarly reduced end-joining. DNA end joining reactions were performed using HeLa extract depleted either with anti-RECQ1 polyclonal antibody (upper panel) or with the anti-Ku70/80 monoclonal antibody (lower panel) as described. Mock-depleted extracts used preimmune rabbit or mouse IgG as control for RECQ1 or Ku70/80 depletion, respectively. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Inverted image of a typical gel is shown. ImageJ was used to quantitate dimer product in each case, and the average from at least three independent experiments are indicated including SD. Western blot of control un-depleted, mock-depleted and RECQ1 or Ku70/80 depleted extracts used in the end-joining reaction is shown along with a loading control (actin) (right).
    T4 Ligase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 114 article reviews
    Price from $9.99 to $1999.99
    t4 ligase buffer - by Bioz Stars, 2020-07
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    Image Search Results


    Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.

    Journal: PLoS ONE

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    doi: 10.1371/journal.pone.0190062

    Figure Lengend Snippet: Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.

    Article Snippet: Reactions included 1 uM of the ligase and 100 nM of the substrate and T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) or NEBuffer 2 (10 mM Tris pH 7.9 @ 25°C, 50 mM NaCl, 10 mM MgCl2 , 1 mM DTT).

    Techniques: Binding Assay

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Journal: PLoS ONE

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    doi: 10.1371/journal.pone.0190062

    Figure Lengend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Article Snippet: Reactions included 1 uM of the ligase and 100 nM of the substrate and T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) or NEBuffer 2 (10 mM Tris pH 7.9 @ 25°C, 50 mM NaCl, 10 mM MgCl2 , 1 mM DTT).

    Techniques: Ligation, Agarose Gel Electrophoresis, Staining

    Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Journal: PLoS ONE

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    doi: 10.1371/journal.pone.0190062

    Figure Lengend Snippet: Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Article Snippet: Reactions included 1 uM of the ligase and 100 nM of the substrate and T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) or NEBuffer 2 (10 mM Tris pH 7.9 @ 25°C, 50 mM NaCl, 10 mM MgCl2 , 1 mM DTT).

    Techniques: Electrophoresis, Produced, Ligation, Standard Deviation

    Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Journal: PLoS ONE

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    doi: 10.1371/journal.pone.0190062

    Figure Lengend Snippet: Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Article Snippet: Reactions included 1 uM of the ligase and 100 nM of the substrate and T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) or NEBuffer 2 (10 mM Tris pH 7.9 @ 25°C, 50 mM NaCl, 10 mM MgCl2 , 1 mM DTT).

    Techniques: Ligation, Produced, Standard Deviation

    YY1 Can Enhance DNA Interactions In Vitro (A and D) Models depicting the in vitro DNA circularization assays used to detect the ability of YY1 to enhance DNA looping interactions with no motif control (A) or competitor DNA control (D). (B and E) Results of the in vitro DNA circularization assay visualized by gel electrophoresis with no motif control (B) or competitor DNA control (E). The dominant lower band reflects the starting linear DNA template, while the upper band corresponds to the circularized DNA ligation product. (C and F) Quantifications of DNA template circularization as a function of incubation time with T4 DNA ligase for no motif control (C) or competitor DNA control (F). Values correspond to the percent of DNA template that is circularized and represents the mean and SD of four experiments. .

    Journal: Cell

    Article Title: YY1 Is a Structural Regulator of Enhancer-Promoter Loops

    doi: 10.1016/j.cell.2017.11.008

    Figure Lengend Snippet: YY1 Can Enhance DNA Interactions In Vitro (A and D) Models depicting the in vitro DNA circularization assays used to detect the ability of YY1 to enhance DNA looping interactions with no motif control (A) or competitor DNA control (D). (B and E) Results of the in vitro DNA circularization assay visualized by gel electrophoresis with no motif control (B) or competitor DNA control (E). The dominant lower band reflects the starting linear DNA template, while the upper band corresponds to the circularized DNA ligation product. (C and F) Quantifications of DNA template circularization as a function of incubation time with T4 DNA ligase for no motif control (C) or competitor DNA control (F). Values correspond to the percent of DNA template that is circularized and represents the mean and SD of four experiments. .

    Article Snippet: Reactions were prepared on ice in 66 μL with the following components: BSA control: 0.25 nM DNA, 1× T4 DNA ligase buffer (NEB B0202S), H2 O 0.12 μg/μL of BSA.

    Techniques: In Vitro, Nucleic Acid Electrophoresis, DNA Ligation, Incubation

    Nucleoprotein filament dynamics on low sequence complexity ssDNA curtains. (A) Sequences of the two ssDNA oligonucleotides used for rolling circle replication. (B) Schematic of rolling circle replication (RCR) reaction. T4 DNA ligase ligates the template oligo to form a contiguous template strand. Next, phi29 DNA polymerase catalyzes the synthesis of long ssDNA molecules. (C) Agarose gel of several time points along the RCR synthesis reaction. The primer oligonucleotide was 32 P labeled on the 5 ′ -terminus phosphate ( gold star ). (D) Wide-field image of a microfabricated barrier set with double-tethered ssDNA curtains coated with RPA-TagRFP ( magenta ). Arrows and circles denote chromium barriers and pedestals, respectively. (E) Illustration and kymograph showing a single ssDNA molecule coated with ATTO488-RAD51(C319S) ( green ) replaced by RPA-TagRFP ( magenta ). Yellow dashed line denotes the injection of RPA–TagRFP into the flowcell. Buffer controls indicate when the buffer flow was toggled off and on to show that the florescent proteins retract to the Cr barriers simultaneously with the ssDNA molecule. This indicates that RAD51 and RPA are on the ssDNA molecule. Panel A: Adapted from Lee, K. S., Marciel, A. B., Kozlov, A. G., Schroeder, C. M., Lohman, T. M., Ha, T. (2014). Ultrafast redistribution of E. coli SSB along long single-stranded DNA via intersegment transfer. Journal of Molecular Biology, 426 , 2413 – 2421.

    Journal: Methods in enzymology

    Article Title: Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination

    doi: 10.1016/bs.mie.2017.03.011

    Figure Lengend Snippet: Nucleoprotein filament dynamics on low sequence complexity ssDNA curtains. (A) Sequences of the two ssDNA oligonucleotides used for rolling circle replication. (B) Schematic of rolling circle replication (RCR) reaction. T4 DNA ligase ligates the template oligo to form a contiguous template strand. Next, phi29 DNA polymerase catalyzes the synthesis of long ssDNA molecules. (C) Agarose gel of several time points along the RCR synthesis reaction. The primer oligonucleotide was 32 P labeled on the 5 ′ -terminus phosphate ( gold star ). (D) Wide-field image of a microfabricated barrier set with double-tethered ssDNA curtains coated with RPA-TagRFP ( magenta ). Arrows and circles denote chromium barriers and pedestals, respectively. (E) Illustration and kymograph showing a single ssDNA molecule coated with ATTO488-RAD51(C319S) ( green ) replaced by RPA-TagRFP ( magenta ). Yellow dashed line denotes the injection of RPA–TagRFP into the flowcell. Buffer controls indicate when the buffer flow was toggled off and on to show that the florescent proteins retract to the Cr barriers simultaneously with the ssDNA molecule. This indicates that RAD51 and RPA are on the ssDNA molecule. Panel A: Adapted from Lee, K. S., Marciel, A. B., Kozlov, A. G., Schroeder, C. M., Lohman, T. M., Ha, T. (2014). Ultrafast redistribution of E. coli SSB along long single-stranded DNA via intersegment transfer. Journal of Molecular Biology, 426 , 2413 – 2421.

    Article Snippet: TE buffer: 10m M Tris–HCl [pH 8.0]; 0.1m M EDTA RAD51 buffer: 40m M Tris–HCl [pH 8.0]; 1m M MgCl2 ; 5m M CaCl2 ; 100m M KCl; 1m M DTT; 1m M ATP; 0.2 mgmL−1 BSA; 1m M Trolox (Sigma-Aldrich); 1.0% glucose (w/v); 500units catalase (Sigma-Aldrich); 70units glucose oxidase (Sigma-Aldrich) 10× T4 DNA ligase reaction buffer (B0202S; NEB) T4 DNA ligase (M0202; NEB) Primer oligo (/Biosg/TC TCC TCC TTC T—HPLC purified; Integrated DNA Technologies) Template oligo (/5Phos/AG GAG AAA AAG AAA AAA AGA AAA GAA GG—PAGE purified; Integrated DNA Technologies) Nuclease-free water BSA, Molecular Biology Grade (B9000S; NEB) Thermocycler (Mastercycler pro S; Eppendorf ) 10× phi29 DNA polymerase reaction buffer (B0269S; NEB) phi29 DNA polymerase (homemade 5 μ M stock) Deoxynucleotide (dNTP) solution set (N0446S; NEB)

    Techniques: Sequencing, Agarose Gel Electrophoresis, Labeling, Recombinase Polymerase Amplification, Injection, Flow Cytometry

    RECQ1 modulates DNA end-joining. A. RECQ1 affects DNA end-joining by T4 ligase. Comparison of ligation products of 5′-cohesive (left panel) or blunt (right panel) ended linear DNA after incubation with T4 ligase alone, or after pre-incubation with the increasing amount of RECQ1 (0–260 ng) or Ku70/80 (0–400 ng) as described in materials and methods. IgG (1µg) was included as unrelated protein in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. O.C., open circular. DNA size marker is shown in leftmost lane. B. RECQ1 antibody interferes with cell free extract mediated end-joining in vitro . The effect of antibodies against human RECQ1 (1.5 and 3 µg) or Ku70/80 (1 and 2 µg) was tested in DNA end-joining reactions assembled with 5 µg HeLa cell free extract. Antibodies at the indicated amounts were incubated with cell free extract for 10 min at room temperature in NHEJ buffer without DNA and ATP. Subsequently, EcoRI-linearized pUC19 DNA and ATP were added to start the reaction followed by 2 h incubation at room temperature. Reaction products were purified and analyzed by SYBR Gold staining after resolution on agarose gel. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. C. Immunodepletion of RECQ1 or Ku70/80 from cell extracts results in similarly reduced end-joining. DNA end joining reactions were performed using HeLa extract depleted either with anti-RECQ1 polyclonal antibody (upper panel) or with the anti-Ku70/80 monoclonal antibody (lower panel) as described. Mock-depleted extracts used preimmune rabbit or mouse IgG as control for RECQ1 or Ku70/80 depletion, respectively. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Inverted image of a typical gel is shown. ImageJ was used to quantitate dimer product in each case, and the average from at least three independent experiments are indicated including SD. Western blot of control un-depleted, mock-depleted and RECQ1 or Ku70/80 depleted extracts used in the end-joining reaction is shown along with a loading control (actin) (right).

    Journal: PLoS ONE

    Article Title: Human RECQ1 Interacts with Ku70/80 and Modulates DNA End-Joining of Double-Strand Breaks

    doi: 10.1371/journal.pone.0062481

    Figure Lengend Snippet: RECQ1 modulates DNA end-joining. A. RECQ1 affects DNA end-joining by T4 ligase. Comparison of ligation products of 5′-cohesive (left panel) or blunt (right panel) ended linear DNA after incubation with T4 ligase alone, or after pre-incubation with the increasing amount of RECQ1 (0–260 ng) or Ku70/80 (0–400 ng) as described in materials and methods. IgG (1µg) was included as unrelated protein in a control reaction. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. O.C., open circular. DNA size marker is shown in leftmost lane. B. RECQ1 antibody interferes with cell free extract mediated end-joining in vitro . The effect of antibodies against human RECQ1 (1.5 and 3 µg) or Ku70/80 (1 and 2 µg) was tested in DNA end-joining reactions assembled with 5 µg HeLa cell free extract. Antibodies at the indicated amounts were incubated with cell free extract for 10 min at room temperature in NHEJ buffer without DNA and ATP. Subsequently, EcoRI-linearized pUC19 DNA and ATP were added to start the reaction followed by 2 h incubation at room temperature. Reaction products were purified and analyzed by SYBR Gold staining after resolution on agarose gel. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. C. Immunodepletion of RECQ1 or Ku70/80 from cell extracts results in similarly reduced end-joining. DNA end joining reactions were performed using HeLa extract depleted either with anti-RECQ1 polyclonal antibody (upper panel) or with the anti-Ku70/80 monoclonal antibody (lower panel) as described. Mock-depleted extracts used preimmune rabbit or mouse IgG as control for RECQ1 or Ku70/80 depletion, respectively. Linear substrate DNA is indicated as monomer, and the end-joined products corresponding to dimer and trimer are indicated. Inverted image of a typical gel is shown. ImageJ was used to quantitate dimer product in each case, and the average from at least three independent experiments are indicated including SD. Western blot of control un-depleted, mock-depleted and RECQ1 or Ku70/80 depleted extracts used in the end-joining reaction is shown along with a loading control (actin) (right).

    Article Snippet: T4 Ligase Assay Substrate DNA (100 ng) was mixed in 10 µl of 1x T4 ligase buffer supplemented with 0.2 mg/ml BSA, with indicated amounts of RECQ1 or Ku70/80 protein, pre-incubated for 10 min at room temperature followed by addition of T4 ligase (200 U, NEB) in 10 µl 1x T4 ligase/BSA buffer.

    Techniques: Ligation, Incubation, Marker, In Vitro, Non-Homologous End Joining, Purification, Staining, Agarose Gel Electrophoresis, Western Blot