t4 ligase buffer  (New England Biolabs)


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    Name:
    T4 DNA Ligase Reaction Buffer
    Description:
    T4 DNA Ligase Reaction Buffer 6 0 ml
    Catalog Number:
    b0202s
    Price:
    24
    Size:
    6 0 ml
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    Buffers
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    Structured Review

    New England Biolabs t4 ligase buffer
    T4 DNA Ligase Reaction Buffer
    T4 DNA Ligase Reaction Buffer 6 0 ml
    https://www.bioz.com/result/t4 ligase buffer/product/New England Biolabs
    Average 99 stars, based on 114 article reviews
    Price from $9.99 to $1999.99
    t4 ligase buffer - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Human RECQ1 Interacts with Ku70/80 and Modulates DNA End-Joining of Double-Strand Breaks"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0062481

    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).
    Figure Legend 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).

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

    2) Product Images from "Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro"

    Article Title: Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro

    Journal: Nucleic Acids Research

    doi:

    Strategy for constructing nicked heteroduplexes. A mismatch-containing oligonucleotide duplex (Fig. 1) is ligated into a template plasmid molecule (1). Linearization of the plasmid (2) in the presence of the heteroduplex oligo, T4 ligase and restriction enzyme ( Bam HI) allows ligation of the small fragments onto each DNA end as a dead-end complex (3), because the Bam HI site is eliminated. Re-ligation of Bam HI-generated plasmid ends yields a molecule competent for a second digestion, returning them to the substrate pool. In the next step, digestion with Eco RI removes one ligation product and generates a ligation-competent DNA end (4). After removal of the smaller fragment, an intramolecular ligation reaction generates the nicked circular product (5). Unwanted linear molecules are removed by digestion with Exonuclease V (Materials and Methods).
    Figure Legend Snippet: Strategy for constructing nicked heteroduplexes. A mismatch-containing oligonucleotide duplex (Fig. 1) is ligated into a template plasmid molecule (1). Linearization of the plasmid (2) in the presence of the heteroduplex oligo, T4 ligase and restriction enzyme ( Bam HI) allows ligation of the small fragments onto each DNA end as a dead-end complex (3), because the Bam HI site is eliminated. Re-ligation of Bam HI-generated plasmid ends yields a molecule competent for a second digestion, returning them to the substrate pool. In the next step, digestion with Eco RI removes one ligation product and generates a ligation-competent DNA end (4). After removal of the smaller fragment, an intramolecular ligation reaction generates the nicked circular product (5). Unwanted linear molecules are removed by digestion with Exonuclease V (Materials and Methods).

    Techniques Used: Plasmid Preparation, Ligation, Generated

    3) Product Images from "Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro"

    Article Title: Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro

    Journal: Nucleic Acids Research

    doi:

    Strategy for constructing nicked heteroduplexes. A mismatch-containing oligonucleotide duplex (Fig. 1) is ligated into a template plasmid molecule (1). Linearization of the plasmid (2) in the presence of the heteroduplex oligo, T4 ligase and restriction enzyme ( Bam HI) allows ligation of the small fragments onto each DNA end as a dead-end complex (3), because the Bam HI site is eliminated. Re-ligation of Bam HI-generated plasmid ends yields a molecule competent for a second digestion, returning them to the substrate pool. In the next step, digestion with Eco RI removes one ligation product and generates a ligation-competent DNA end (4). After removal of the smaller fragment, an intramolecular ligation reaction generates the nicked circular product (5). Unwanted linear molecules are removed by digestion with Exonuclease V (Materials and Methods).
    Figure Legend Snippet: Strategy for constructing nicked heteroduplexes. A mismatch-containing oligonucleotide duplex (Fig. 1) is ligated into a template plasmid molecule (1). Linearization of the plasmid (2) in the presence of the heteroduplex oligo, T4 ligase and restriction enzyme ( Bam HI) allows ligation of the small fragments onto each DNA end as a dead-end complex (3), because the Bam HI site is eliminated. Re-ligation of Bam HI-generated plasmid ends yields a molecule competent for a second digestion, returning them to the substrate pool. In the next step, digestion with Eco RI removes one ligation product and generates a ligation-competent DNA end (4). After removal of the smaller fragment, an intramolecular ligation reaction generates the nicked circular product (5). Unwanted linear molecules are removed by digestion with Exonuclease V (Materials and Methods).

    Techniques Used: Plasmid Preparation, Ligation, Generated

    4) Product Images from "A simple and efficient cloning system for CRISPR/Cas9-mediated genome editing in rice"

    Article Title: A simple and efficient cloning system for CRISPR/Cas9-mediated genome editing in rice

    Journal: PeerJ

    doi: 10.7717/peerj.8491

    Workflow for constructing expression clone containing two target-sgRNA expression cassettes with Golden Gate clone. Primers containing adaptors for Golden Gate cloning (OJH307 and OJH308) were used in the amplification with PJG090 as the template. The reagents were recommended as following: one ul of PCR product, 50 ng of PJG112, one ul of Cutsmart Buffer (NEB), 0.4 ul of T4 ligase buffer (NEB), 5 U of Bsa I (NEB), 20 U of T4 DNA ligase (NEB) and add ddH 2 O to 10 ul. The reaction was incubated for 20–25 cycles (37 °C 2 min, 20 °C 5 min), followed by incubation at 50 °C and 80 °C for 5 min, respectively. Subsequently, one ul of the product was introduced into Trans T1 competent cells. Positive clones were identified by clone PCR and sequenced.
    Figure Legend Snippet: Workflow for constructing expression clone containing two target-sgRNA expression cassettes with Golden Gate clone. Primers containing adaptors for Golden Gate cloning (OJH307 and OJH308) were used in the amplification with PJG090 as the template. The reagents were recommended as following: one ul of PCR product, 50 ng of PJG112, one ul of Cutsmart Buffer (NEB), 0.4 ul of T4 ligase buffer (NEB), 5 U of Bsa I (NEB), 20 U of T4 DNA ligase (NEB) and add ddH 2 O to 10 ul. The reaction was incubated for 20–25 cycles (37 °C 2 min, 20 °C 5 min), followed by incubation at 50 °C and 80 °C for 5 min, respectively. Subsequently, one ul of the product was introduced into Trans T1 competent cells. Positive clones were identified by clone PCR and sequenced.

    Techniques Used: Expressing, Clone Assay, Amplification, Polymerase Chain Reaction, Incubation

    Related Articles

    Amplification:

    Article Title: Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples
    Article Snippet: .. End-repair without T4 DNA Polymerase: for treatments −/+ and −/−, an end-repair mastermix was made by combining 4 μl T4 DNA ligase reaction buffer (New England Biolabs, NEB, Ipswich, Massachusetts, US), 0.5 μl dATP (10mM) (Thermo-Fisher), 2 μl reaction booster mix (consisting of 25 % PEG-4000 (Sigma Aldrich, 50%), 2 mg/ml BSA (Thermo-Fisher) and 400 mM NaCl) , 2 μl T4 PNK (NEB, cat#M0201S, 10 U/μl) and 1.5 μl Klenow Fragment (3’- > 5’ exo-) (NEB, cat#M0212S, 5 U/μl) per amplicon pool reaction. .. Ten μl of this mastermix was then added to each 30 μl amplicon pool, mixed well by pipetting and incubated for 30 minutes at 37°C followed by 30 minutes at 65°C and finally cooled to 4°C.

    Ligation:

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases
    Article Snippet: .. 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 MgCl2 ) 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)). (TIF) Click here for additional data file. .. Fluorescence anisotropy DNA binding curves.

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases
    Article Snippet: .. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer + 150 mM NaCl (50 mM Tris-HCl pH 7.5 @ 25°C, 150 mM NaCl, 1 mM ATP and 10 mM MgCl2 ) or NEBNext® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2 , 1 mM DTT, 150 mM NaCl, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). ..

    Article Title: Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro
    Article Snippet: .. A final ligation, which creates the nicked circular substrate, was carried out at a DNA concentration of 10 ng/µl in T4 ligase buffer supplemented with 50 mM NaCl, Eco RI (2 U/µg plasmid), T4 ligase (100 U/µg plasmid) and 100 µg/ml bovine serum albumin. ..

    Purification:

    Article Title: Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination
    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) .. Prepare a 49 μL ligation reaction containing: (i) 5 μL 10× T4 ligase reaction buffer; (ii) 2 μL template oligo (10 μ M stock in TE buffer); (iii) 1.8 μL primer oligo (10 μ M stock in TE buffer); and (iv) 40.2 μL nuclease-free water.

    Concentration Assay:

    Article Title: Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro
    Article Snippet: .. A final ligation, which creates the nicked circular substrate, was carried out at a DNA concentration of 10 ng/µl in T4 ligase buffer supplemented with 50 mM NaCl, Eco RI (2 U/µg plasmid), T4 ligase (100 U/µg plasmid) and 100 µg/ml bovine serum albumin. ..

    Incubation:

    Article Title: Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro
    Article Snippet: .. The reaction was incubated for 10 min at 37°C in T4 ligase buffer (New England Biolabs) containing 100 µg/ml bovine serum albumin, 75 mM KCl and the heteroduplex oligo recovered after Dpn II digestion (estimated to be a ∼100-fold molar excess over the plasmid ends). .. The reaction was then cooled on ice, whereupon 100 cohesive end ligation units of T4 ligase per microgram of plasmid (1500 U in this example) were added and the reaction incubated at 16°C overnight.

    Plasmid Preparation:

    Article Title: Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro
    Article Snippet: .. A final ligation, which creates the nicked circular substrate, was carried out at a DNA concentration of 10 ng/µl in T4 ligase buffer supplemented with 50 mM NaCl, Eco RI (2 U/µg plasmid), T4 ligase (100 U/µg plasmid) and 100 µg/ml bovine serum albumin. ..

    Article Title: Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro
    Article Snippet: .. The reaction was incubated for 10 min at 37°C in T4 ligase buffer (New England Biolabs) containing 100 µg/ml bovine serum albumin, 75 mM KCl and the heteroduplex oligo recovered after Dpn II digestion (estimated to be a ∼100-fold molar excess over the plasmid ends). .. The reaction was then cooled on ice, whereupon 100 cohesive end ligation units of T4 ligase per microgram of plasmid (1500 U in this example) were added and the reaction incubated at 16°C overnight.

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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
    Average 99 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase reaction buffer - by Bioz Stars, 2020-07
    99/100 stars
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    99
    New England Biolabs t4 rna ligase buffer
    DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using <t>T4</t> RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.
    T4 Rna Ligase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase buffer/product/New England Biolabs
    Average 99 stars, based on 41 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase buffer - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    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

    DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using T4 RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.

    Journal: Genome Research

    Article Title: Strand-specific deep sequencing of the transcriptome

    doi: 10.1101/gr.094318.109

    Figure Lengend Snippet: DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using T4 RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.

    Article Snippet: We incubated the following reaction mixture for 30 min at 37°C: 10 μL of sample, 1 μL of 10× T4 RNA ligase buffer (as fresh ATP supply), 10 U of polynucleotide kinase (New England BioLabs), 3 μL of RNase free water.

    Techniques: De-Phosphorylation Assay, Ligation, Polyacrylamide Gel Electrophoresis, Selection, Amplification, Polymerase Chain Reaction, Sequencing