t4 dna ligase reaction buffer  (New England Biolabs)


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    Name:
    T4 DNA Ligase Reaction Buffer
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    T4 DNA Ligase Reaction Buffer 6 0 ml
    Catalog Number:
    B0202S
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    6 0 ml
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    New England Biolabs t4 dna ligase reaction buffer
    T4 DNA Ligase Reaction Buffer
    T4 DNA Ligase Reaction Buffer 6 0 ml
    https://www.bioz.com/result/t4 dna ligase reaction buffer/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
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    t4 dna ligase reaction buffer - by Bioz Stars, 2021-05
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    Images

    1) Product Images from "Assessing Protein Dynamics on Low-Complexity Single-Stranded DNA Curtains"

    Article Title: Assessing Protein Dynamics on Low-Complexity Single-Stranded DNA Curtains

    Journal: Langmuir : the ACS journal of surfaces and colloids

    doi: 10.1021/acs.langmuir.8b01812

    Assembly of low-complexity ssDNA curtains. (A) A phosphorylated template (black) and a biotinylated primer (green) are annealed and treated with T4 DNA ligase to make minicircles. Low-complexity ssDNA composed solely of thymidine and cytidine is synthesized via rolling circle replication by phi29 DNAP. (B) Low-complexity ssDNA curtains with fluorescent end labeling. The 3′ end of the ssDNA was labeled with a fluorescent antibody. (C) RPA-GFP (green)-coated ssDNA with fluorescent end labeling (magenta). (D) Kymograph of a representative ssDNA in panel (C) with buffer flow on and off, indicating that the ssDNA is anchored to the surface via the 5′-biotin tether.
    Figure Legend Snippet: Assembly of low-complexity ssDNA curtains. (A) A phosphorylated template (black) and a biotinylated primer (green) are annealed and treated with T4 DNA ligase to make minicircles. Low-complexity ssDNA composed solely of thymidine and cytidine is synthesized via rolling circle replication by phi29 DNAP. (B) Low-complexity ssDNA curtains with fluorescent end labeling. The 3′ end of the ssDNA was labeled with a fluorescent antibody. (C) RPA-GFP (green)-coated ssDNA with fluorescent end labeling (magenta). (D) Kymograph of a representative ssDNA in panel (C) with buffer flow on and off, indicating that the ssDNA is anchored to the surface via the 5′-biotin tether.

    Techniques Used: Synthesized, End Labeling, Labeling, Recombinase Polymerase Amplification, Flow Cytometry

    2) Product Images from "YY1 Is a Structural Regulator of Enhancer-Promoter Loops"

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

    Journal: Cell

    doi: 10.1016/j.cell.2017.11.008

    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. .
    Figure Legend 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. .

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

    3) Product Images from "A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage"

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq756

    Principle, reaction efficiencies and NMR evidence for isotope labeling of each stem-loop of the RsmZ RNA separately. ( a ) Sequence-specific RNase H cleavages to obtain all four isotopically labeled stem-loop fragments. The yields of the cleavage reactions before HPLC purification are indicated, the values in brackets are expressing the yield after purification. The different stem-loops are colored (SL1: magenta, SL2: green, SL3: orange, SL4: cyan). ( b ) Splinted T4 DNA ligase mediated ligations of isotope labeled (in color) and unlabeled (in black) fragments. The unlabeled fragments were obtained in a similar way as the labeled fragments. ( c ) NMR evidence for the successful segmental isotope labeling of each stem-loop separately. 1 H- 15 N-HSQC NMR spectrum of the uniformly 15 N-labeled RsmZ RNA (left) and overlay of the 1 H- 15 N-HSQC NMR spectra of the four segmentally labeled RsmZ RNAs with each stem-loop labeled separately (right). The spectra were recorded on a Bruker 600 MHz spectrometer at 10°C.
    Figure Legend Snippet: Principle, reaction efficiencies and NMR evidence for isotope labeling of each stem-loop of the RsmZ RNA separately. ( a ) Sequence-specific RNase H cleavages to obtain all four isotopically labeled stem-loop fragments. The yields of the cleavage reactions before HPLC purification are indicated, the values in brackets are expressing the yield after purification. The different stem-loops are colored (SL1: magenta, SL2: green, SL3: orange, SL4: cyan). ( b ) Splinted T4 DNA ligase mediated ligations of isotope labeled (in color) and unlabeled (in black) fragments. The unlabeled fragments were obtained in a similar way as the labeled fragments. ( c ) NMR evidence for the successful segmental isotope labeling of each stem-loop separately. 1 H- 15 N-HSQC NMR spectrum of the uniformly 15 N-labeled RsmZ RNA (left) and overlay of the 1 H- 15 N-HSQC NMR spectra of the four segmentally labeled RsmZ RNAs with each stem-loop labeled separately (right). The spectra were recorded on a Bruker 600 MHz spectrometer at 10°C.

    Techniques Used: Nuclear Magnetic Resonance, Labeling, Sequencing, High Performance Liquid Chromatography, Purification, Expressing

    4) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Ligation, Produced, Standard Deviation

    5) 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

    6) Product Images from "Mechanical properties of DNA-like polymers"

    Article Title: Mechanical properties of DNA-like polymers

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt808

    Example measurement of mechanical properties. ( A ) Cyclization time course for 207-bp DNA-like polymer 5 (pJ1744). DNA ligase-catalyzed cyclization reaction was performed at ∼22°C with 1 nM DNA restriction fragment, T4 DNA ligation buffer (50 mM Tris–HCl, pH 7.5, 10 mM MgCl 2 , 1 mM ATP, 10 mM dithiothreitol) and a final concentration of 100 U/ml T4 DNA ligase. Aliquots (10 µl) were removed at 1–15 min time points, quenched by addition of EDTA to 20 mM and then analyzed by electrophoresis through 5% native polyacrylamide gels in 0.5× TBE buffer (50 mM Tris base, 55 mM boric acid and 1 mM EDTA, pH 8.3). Gel lanes contains Invitrogen 100 bp DNA ladder (M), linear monomer without ligase (0) and increasing 1-min time points of the ligation reaction ( 1–15 ) showing the evolution of linear monomer ( M ), linear dimer ( D ), circular monomer ( C M ) and circular dimer ( C D ). Nearest molecular weight bands are indicated. ( B ) Cyclization kinetics analysis for 207-bp DNA-like polymer 5 (pJ1744). Fitting of data in (A) determines the J -factor, as previously described ( 30 ) (see also Supplementary Data S3 ). ( C ) WLC analysis for DNA-like polymer 5 . Fit of experimental J -factor data using the WLC model. ( D ). Monte Carlo estimation of uncertainty. Fit of simulated J -factor data based on (C) using the WLC model.
    Figure Legend Snippet: Example measurement of mechanical properties. ( A ) Cyclization time course for 207-bp DNA-like polymer 5 (pJ1744). DNA ligase-catalyzed cyclization reaction was performed at ∼22°C with 1 nM DNA restriction fragment, T4 DNA ligation buffer (50 mM Tris–HCl, pH 7.5, 10 mM MgCl 2 , 1 mM ATP, 10 mM dithiothreitol) and a final concentration of 100 U/ml T4 DNA ligase. Aliquots (10 µl) were removed at 1–15 min time points, quenched by addition of EDTA to 20 mM and then analyzed by electrophoresis through 5% native polyacrylamide gels in 0.5× TBE buffer (50 mM Tris base, 55 mM boric acid and 1 mM EDTA, pH 8.3). Gel lanes contains Invitrogen 100 bp DNA ladder (M), linear monomer without ligase (0) and increasing 1-min time points of the ligation reaction ( 1–15 ) showing the evolution of linear monomer ( M ), linear dimer ( D ), circular monomer ( C M ) and circular dimer ( C D ). Nearest molecular weight bands are indicated. ( B ) Cyclization kinetics analysis for 207-bp DNA-like polymer 5 (pJ1744). Fitting of data in (A) determines the J -factor, as previously described ( 30 ) (see also Supplementary Data S3 ). ( C ) WLC analysis for DNA-like polymer 5 . Fit of experimental J -factor data using the WLC model. ( D ). Monte Carlo estimation of uncertainty. Fit of simulated J -factor data based on (C) using the WLC model.

    Techniques Used: DNA Ligation, Concentration Assay, Electrophoresis, Ligation, Molecular Weight

    7) Product Images from "Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination"

    Article Title: Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination

    Journal: Nature Communications

    doi: 10.1038/s41467-021-21275-4

    Characterization of various genomic DNA digestion/DNA assembly combinations in the CAPTURE method. a Schematics of T4 DNA polymerase exo + fill-in DNA assembly. In step 1, DNA molecules ends are chewed back by T4 DNA polymerase to create ssDNA overhangs. The reaction mixture’s temperature is increased to 75 °C to inactivate T4 DNA polymerase and potentially remove ssDNA secondary structures. Temperature is then decreased to 50 °C to allow for ssDNA overhang hybridization. In step 2, by addition of fresh T4 DNA polymerase, and dNTPs, DNA gaps in the hybridized DNA molecule are filled. E. coli DNA ligase is then used to ligate the nicks and produce the final assembly product. b Comparison of different digestion/DNA assembly combinations in cloning four high GC-content BGCs from Actinomycetes. The Fn Cas12a/T4 exo + fill-in strategy showed ~100% cloning efficiency for all four target BGCs. RE: restriction enzymes. For each cloning experiment, at least seven colonies were selected and the purified plasmids from each colony were analyzed by restriction digestion. The cloning efficiencies were calculated as the ratio of correct colonies to the total number of checked colonies. Each experiment was performed in three biological replicates and data are presented as mean values ± standard error (SEM). c Summary of results for cloning uncharacterized BGCs using CAPTURE. BGCs ranging from 10 to 113 kb can be robustly cloned using the CAPTURE method at close to 100% efficiency regardless of their GC-content. Source data are provided as a Source Data file.
    Figure Legend Snippet: Characterization of various genomic DNA digestion/DNA assembly combinations in the CAPTURE method. a Schematics of T4 DNA polymerase exo + fill-in DNA assembly. In step 1, DNA molecules ends are chewed back by T4 DNA polymerase to create ssDNA overhangs. The reaction mixture’s temperature is increased to 75 °C to inactivate T4 DNA polymerase and potentially remove ssDNA secondary structures. Temperature is then decreased to 50 °C to allow for ssDNA overhang hybridization. In step 2, by addition of fresh T4 DNA polymerase, and dNTPs, DNA gaps in the hybridized DNA molecule are filled. E. coli DNA ligase is then used to ligate the nicks and produce the final assembly product. b Comparison of different digestion/DNA assembly combinations in cloning four high GC-content BGCs from Actinomycetes. The Fn Cas12a/T4 exo + fill-in strategy showed ~100% cloning efficiency for all four target BGCs. RE: restriction enzymes. For each cloning experiment, at least seven colonies were selected and the purified plasmids from each colony were analyzed by restriction digestion. The cloning efficiencies were calculated as the ratio of correct colonies to the total number of checked colonies. Each experiment was performed in three biological replicates and data are presented as mean values ± standard error (SEM). c Summary of results for cloning uncharacterized BGCs using CAPTURE. BGCs ranging from 10 to 113 kb can be robustly cloned using the CAPTURE method at close to 100% efficiency regardless of their GC-content. Source data are provided as a Source Data file.

    Techniques Used: Hybridization, Clone Assay, Purification

    Development of the CAPTURE method. a Overview of the workflow. In the first step, purified genomic DNA is digested by Cas12a enzyme to release the target BGC fragment. In the second step, digestion products are mixed with two DNA receivers containing lox P sites at their ends. The target BGC fragment and DNA receivers are assembled together using T4 DNA polymerase exo + fill-in DNA assembly. In the final step, the assembly mixture is transformed into E. coli cells harboring a circularization helper plasmid. The linear DNA is able to circularize in vivo by Cre- lox recombination. b DNA map of helper plasmid pBE14. tcr : tetracycline resistance marker; araBAD : L-arabinose inducible promoter and its regulator; gam : phage lambda Red gam gene; pSC101: temperature-sensitive origin of replication; recA1 : mutated E. coli recA gene to increase transformation efficiency. c Comparison of recombination frequency between Flp (pBE11) and Cre (pBE12) helper plasmids. -: without L-arabinose induction, +: with L-arabinose induction. Recombination frequencies were calculated based on the ratio of white colonies to the total number of acquired colonies. d Linear DNA transformation efficiency for E. coli cells harboring pBE11 (Flp), pBE12 (Cre), pBE14 (Cre and recA1) helper plasmids. Both pBE12 and pBE14 E. coli cells exhibited transformation efficiencies similar to circular DNA. e Comparison of in vitro versus in vivo circularization for two large (50 kb, 73 kb) linear DNA molecules. In vivo circularization showed ~33-fold and 150-fold higher frequency than in vitro circularization for 50 kb and 73 kb molecules, respectively. Circularization frequencies were calculated based on the number of colonies acquired for each circularization experiment in comparison to the number of colonies acquired after transformation of the original circular DNA (see Methods for full description). Each experiment was performed in three biological replicates and data are presented as mean values ± standard deviation (SD). Source data are provided as a Source Data file.
    Figure Legend Snippet: Development of the CAPTURE method. a Overview of the workflow. In the first step, purified genomic DNA is digested by Cas12a enzyme to release the target BGC fragment. In the second step, digestion products are mixed with two DNA receivers containing lox P sites at their ends. The target BGC fragment and DNA receivers are assembled together using T4 DNA polymerase exo + fill-in DNA assembly. In the final step, the assembly mixture is transformed into E. coli cells harboring a circularization helper plasmid. The linear DNA is able to circularize in vivo by Cre- lox recombination. b DNA map of helper plasmid pBE14. tcr : tetracycline resistance marker; araBAD : L-arabinose inducible promoter and its regulator; gam : phage lambda Red gam gene; pSC101: temperature-sensitive origin of replication; recA1 : mutated E. coli recA gene to increase transformation efficiency. c Comparison of recombination frequency between Flp (pBE11) and Cre (pBE12) helper plasmids. -: without L-arabinose induction, +: with L-arabinose induction. Recombination frequencies were calculated based on the ratio of white colonies to the total number of acquired colonies. d Linear DNA transformation efficiency for E. coli cells harboring pBE11 (Flp), pBE12 (Cre), pBE14 (Cre and recA1) helper plasmids. Both pBE12 and pBE14 E. coli cells exhibited transformation efficiencies similar to circular DNA. e Comparison of in vitro versus in vivo circularization for two large (50 kb, 73 kb) linear DNA molecules. In vivo circularization showed ~33-fold and 150-fold higher frequency than in vitro circularization for 50 kb and 73 kb molecules, respectively. Circularization frequencies were calculated based on the number of colonies acquired for each circularization experiment in comparison to the number of colonies acquired after transformation of the original circular DNA (see Methods for full description). Each experiment was performed in three biological replicates and data are presented as mean values ± standard deviation (SD). Source data are provided as a Source Data file.

    Techniques Used: Purification, Transformation Assay, Plasmid Preparation, In Vivo, Marker, In Vitro, Standard Deviation

    8) Product Images from "YY1 Is a Structural Regulator of Enhancer-Promoter Loops"

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

    Journal: Cell

    doi: 10.1016/j.cell.2017.11.008

    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. .
    Figure Legend 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. .

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

    9) 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

    10) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Ligation, Produced, Standard Deviation

    11) Product Images from "A high-throughput expression screening platform to optimize the production of antimicrobial peptides"

    Article Title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-017-0637-5

    Golden Gate assembly of the expression plasmid in a single reaction. Six donor plasmids and one acceptor plasmid were combined to form each expression plasmid by directional cloning in a restriction-ligation step using the endonuclease Bsa I and T4 DNA ligase. The overhangs produced by Bsa I are colored
    Figure Legend Snippet: Golden Gate assembly of the expression plasmid in a single reaction. Six donor plasmids and one acceptor plasmid were combined to form each expression plasmid by directional cloning in a restriction-ligation step using the endonuclease Bsa I and T4 DNA ligase. The overhangs produced by Bsa I are colored

    Techniques Used: Expressing, Plasmid Preparation, Clone Assay, Ligation, Produced

    12) Product Images from "YY1 Is a Structural Regulator of Enhancer-Promoter Loops"

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

    Journal: Cell

    doi: 10.1016/j.cell.2017.11.008

    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. .
    Figure Legend 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. .

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

    13) Product Images from "Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples"

    Article Title: Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples

    Journal: bioRxiv

    doi: 10.1101/2020.01.22.915009

    Average percentage of sequences carrying tag-jumps across amplicon pools built into Illumina libraries with four different library protocol treatments; +/+: T4 DNA polymerase blunt-ending and post-ligation PCR; −/+: no T4 DNA polymerase blunt-ending, with post-ligation PCR; +/−: T4 DNA polymerase blunt-ending and no post-ligation PCR; −/−: no T4 DNA polymerase blunt-ending and no post-ligation PCR (Tagsteady protocol) (n=6 for +/+, −/+, +/−, −/−). To mimic the effect of large amounts of single-stranded DNA generated in the metabarcoding PCR, aliquots of four of the amplicon pools were denatured and subsequently re-hybridized to form double-stranded DNA. These were then built into libraries with the −/− and +/− protocols (n=4 for d+/− and d−/−. Asterisks (*) denotes statistical significant difference between treatments (unpaired t-test, α=0.05).
    Figure Legend Snippet: Average percentage of sequences carrying tag-jumps across amplicon pools built into Illumina libraries with four different library protocol treatments; +/+: T4 DNA polymerase blunt-ending and post-ligation PCR; −/+: no T4 DNA polymerase blunt-ending, with post-ligation PCR; +/−: T4 DNA polymerase blunt-ending and no post-ligation PCR; −/−: no T4 DNA polymerase blunt-ending and no post-ligation PCR (Tagsteady protocol) (n=6 for +/+, −/+, +/−, −/−). To mimic the effect of large amounts of single-stranded DNA generated in the metabarcoding PCR, aliquots of four of the amplicon pools were denatured and subsequently re-hybridized to form double-stranded DNA. These were then built into libraries with the −/− and +/− protocols (n=4 for d+/− and d−/−. Asterisks (*) denotes statistical significant difference between treatments (unpaired t-test, α=0.05).

    Techniques Used: Amplification, Ligation, Polymerase Chain Reaction, Generated

    Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, T4 PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).
    Figure Legend Snippet: Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, T4 PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).

    Techniques Used: Polymerase Chain Reaction, Activity Assay, Sequencing, Ligation, Amplification

    Experimental overview. 1A) Six pools of 5’ twin-tagged amplicons generated with three metabarcoding primer sets were used to assess the effect of blunt-ending and post-ligation PCR on tag-jumps. Each of the six amplicon pools were subjected to four different treatments. 1B) The four treatments represent combinations with and without T4 DNA Polymerase blunt-ending in the end-repair step and 1C) with and without post-ligation PCR. 1D) This resulted in 24 libraries for the 6 amplicon pools, representing four library preparation treatments for each amplicon pool. 2A) To further assess the effect of T4 DNA Polymerase blunt-ending on the prevalence of tag-jumps, we denatured and re-hybridised four amplicon pools (16sMam1/2 and 16sIns1/2). 2B) End-repair was carried out with and without T4 DNA Polymerase blunt-ending and with no post-ligation PCR (2C). 3) Finally, to validate the robustness and stability of the Tagsteady protocol, we applied it to 15 pools of twin-tagged amplicons.
    Figure Legend Snippet: Experimental overview. 1A) Six pools of 5’ twin-tagged amplicons generated with three metabarcoding primer sets were used to assess the effect of blunt-ending and post-ligation PCR on tag-jumps. Each of the six amplicon pools were subjected to four different treatments. 1B) The four treatments represent combinations with and without T4 DNA Polymerase blunt-ending in the end-repair step and 1C) with and without post-ligation PCR. 1D) This resulted in 24 libraries for the 6 amplicon pools, representing four library preparation treatments for each amplicon pool. 2A) To further assess the effect of T4 DNA Polymerase blunt-ending on the prevalence of tag-jumps, we denatured and re-hybridised four amplicon pools (16sMam1/2 and 16sIns1/2). 2B) End-repair was carried out with and without T4 DNA Polymerase blunt-ending and with no post-ligation PCR (2C). 3) Finally, to validate the robustness and stability of the Tagsteady protocol, we applied it to 15 pools of twin-tagged amplicons.

    Techniques Used: Generated, Ligation, Polymerase Chain Reaction, Amplification

    14) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Ligation, Produced, Standard Deviation

    15) Product Images from "Split mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins"

    Article Title: Split mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa270

    Design of bead surface and solid-phase manipulations of DNA. ( A ) Beads were designed to display both azide (labelled ‘N 3 ’) and SpyTag (labelled ‘ST’) moieties (surface modification described in Supplementary Figure S1 ). ( B ) Flow cytometric analysis of beads for fluorescein-derived fluorescence intensity before (grey) and after (black) immobilisation of fluorescein and DBCO-functionalised DNA (top histogram), after Esp3I treatment (2 hours at 37°C) of the DNA-coated beads (middle histogram) and after exposure of Esp3I-treated beads to a fluorescein-labelled DNA duplex that had a 5′-overhang complementary to the 5′-overhang of bead-immobilised DNA, in T4 DNA ligase buffer, with (black) or without (grey) T4 DNA ligase (bottom histogram). Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S4 . ( C ) Schematic overview of on-bead assembly allowing potential saturation of three codons in close proximity. The final, bead-attached DNA assembly is shown at the top of the panel, with the three DNA fragments used in the construction are shown below. Restriction sites are depicted in red, target codons in green and sequences used for hybridisation during ligation in blue. The first, PCR-generated amplicon (frag 3 ) was attached to bead ( via copper-free click chemistry) and digested by Esp3I. DNA on the bead was extended using an oligonucleotide duplex (frag 2 ) carrying a 5′-phosphorylated cohesive end; the sequence used to ensure stability of the duplex (stability stuffer) prior to ligation is indicated in a diagonal pattern. Once this duplex had been appended to the bead by ligation, a new cohesive end was generated (and stability stuffer removed) through BspQI digestion. Finally, another PCR amplicon (frag 1 ), separately prepared with a cohesive end (using BspQI) was ligated to the bead-immobilised DNA. Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S5 . ( D ) Flow cytometric analysis of untreated beads (top trace), beads carrying full length starting template (i.e. with FAM at one end and DBCO at the other, middle trace) and beads having gone through the 3-codon SpliMLiB process described in C. ( E ) Sanger sequencing chromatogram (templated by a PCR amplicon obtained directly from beads) of the exemplary bead-surface assembled construct shown in panel C where codons to be mutated were designed to be in close proximity (bottom). As in panel C, the green coloring refers to mutated positions, while the blue coloring refers to sequences used for ligations.
    Figure Legend Snippet: Design of bead surface and solid-phase manipulations of DNA. ( A ) Beads were designed to display both azide (labelled ‘N 3 ’) and SpyTag (labelled ‘ST’) moieties (surface modification described in Supplementary Figure S1 ). ( B ) Flow cytometric analysis of beads for fluorescein-derived fluorescence intensity before (grey) and after (black) immobilisation of fluorescein and DBCO-functionalised DNA (top histogram), after Esp3I treatment (2 hours at 37°C) of the DNA-coated beads (middle histogram) and after exposure of Esp3I-treated beads to a fluorescein-labelled DNA duplex that had a 5′-overhang complementary to the 5′-overhang of bead-immobilised DNA, in T4 DNA ligase buffer, with (black) or without (grey) T4 DNA ligase (bottom histogram). Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S4 . ( C ) Schematic overview of on-bead assembly allowing potential saturation of three codons in close proximity. The final, bead-attached DNA assembly is shown at the top of the panel, with the three DNA fragments used in the construction are shown below. Restriction sites are depicted in red, target codons in green and sequences used for hybridisation during ligation in blue. The first, PCR-generated amplicon (frag 3 ) was attached to bead ( via copper-free click chemistry) and digested by Esp3I. DNA on the bead was extended using an oligonucleotide duplex (frag 2 ) carrying a 5′-phosphorylated cohesive end; the sequence used to ensure stability of the duplex (stability stuffer) prior to ligation is indicated in a diagonal pattern. Once this duplex had been appended to the bead by ligation, a new cohesive end was generated (and stability stuffer removed) through BspQI digestion. Finally, another PCR amplicon (frag 1 ), separately prepared with a cohesive end (using BspQI) was ligated to the bead-immobilised DNA. Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S5 . ( D ) Flow cytometric analysis of untreated beads (top trace), beads carrying full length starting template (i.e. with FAM at one end and DBCO at the other, middle trace) and beads having gone through the 3-codon SpliMLiB process described in C. ( E ) Sanger sequencing chromatogram (templated by a PCR amplicon obtained directly from beads) of the exemplary bead-surface assembled construct shown in panel C where codons to be mutated were designed to be in close proximity (bottom). As in panel C, the green coloring refers to mutated positions, while the blue coloring refers to sequences used for ligations.

    Techniques Used: Modification, Derivative Assay, Fluorescence, Hybridization, Ligation, Polymerase Chain Reaction, Generated, Amplification, Sequencing, Construct

    16) Product Images from "Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination"

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

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2017.03.011

    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.
    Figure Legend 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.

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

    17) Product Images from "Biochemical Properties of a Decoy Oligodeoxynucleotide Inhibitor of STAT3 Transcription Factor"

    Article Title: Biochemical Properties of a Decoy Oligodeoxynucleotide Inhibitor of STAT3 Transcription Factor

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19061608

    Ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D) is unaffected by biotinylation. ( A ) Structures of parental and biotinylated STAT3 decoy (S3D) and CS3D. ( B ) CS3D and biotinylated CS3D were incubated with T4 DNA ligase overnight, followed by electrophoresis on a urea/polyacrylamide gel and staining with SYBR Gold.
    Figure Legend Snippet: Ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D) is unaffected by biotinylation. ( A ) Structures of parental and biotinylated STAT3 decoy (S3D) and CS3D. ( B ) CS3D and biotinylated CS3D were incubated with T4 DNA ligase overnight, followed by electrophoresis on a urea/polyacrylamide gel and staining with SYBR Gold.

    Techniques Used: Ligation, Incubation, Electrophoresis, Staining

    Efficient ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D). ( A ) Schematic representation of CS3D ligation with T4 DNA ligase. The complementary segments of the single-stranded decoy molecule spontaneously self-anneal. Enzymatic ligation with T4 DNA ligase was used to complete cyclization. ( B ) Incubations were performed in the absence or presence of T4 DNA ligase overnight. Multiple identical ligations ( n = 5) were simultaneously performed. Samples from each reaction were then electrophoresed on a urea/polyacrylamide gel, stained with SYBR Gold, and quantified by densitometry.
    Figure Legend Snippet: Efficient ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D). ( A ) Schematic representation of CS3D ligation with T4 DNA ligase. The complementary segments of the single-stranded decoy molecule spontaneously self-anneal. Enzymatic ligation with T4 DNA ligase was used to complete cyclization. ( B ) Incubations were performed in the absence or presence of T4 DNA ligase overnight. Multiple identical ligations ( n = 5) were simultaneously performed. Samples from each reaction were then electrophoresed on a urea/polyacrylamide gel, stained with SYBR Gold, and quantified by densitometry.

    Techniques Used: Ligation, Staining

    18) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Ligation, Produced, Standard Deviation

    19) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Ligation, Produced, Standard Deviation

    20) 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

    21) Product Images from "Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA"

    Article Title: Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA

    Journal: RNA Biology

    doi: 10.1080/15476286.2020.1777378

    A workflow of sample preparation for determination of positions of DNA cleavage sites produced by Cas nucleases in vitro. The cleaved DNA fragments generated by Cas nuclease during in vitro DNA cleavage reaction (Step I) are blunted using T4 PNK and T4 DNA polymerase (Step II). A-base is added to 3ʹ ends (Step III) for further ligation of Illumina NEBNext sequencing adaptors containing uridine (Step IV). Uridines are cleaved out using the NEB USER enzyme, which combines uracil DNA glycosylase and endonuclease VIII activity. Next, the samples are barcoded to produce DNA libraries ready for high throughput sequencing.
    Figure Legend Snippet: A workflow of sample preparation for determination of positions of DNA cleavage sites produced by Cas nucleases in vitro. The cleaved DNA fragments generated by Cas nuclease during in vitro DNA cleavage reaction (Step I) are blunted using T4 PNK and T4 DNA polymerase (Step II). A-base is added to 3ʹ ends (Step III) for further ligation of Illumina NEBNext sequencing adaptors containing uridine (Step IV). Uridines are cleaved out using the NEB USER enzyme, which combines uracil DNA glycosylase and endonuclease VIII activity. Next, the samples are barcoded to produce DNA libraries ready for high throughput sequencing.

    Techniques Used: Sample Prep, Produced, In Vitro, Generated, Ligation, Sequencing, Activity Assay, Next-Generation Sequencing

    22) Product Images from "Split mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins"

    Article Title: Split mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa270

    Design of bead surface and solid-phase manipulations of DNA. ( A ) Beads were designed to display both azide (labelled ‘N 3 ’) and SpyTag (labelled ‘ST’) moieties (surface modification described in Supplementary Figure S1 ). ( B ) Flow cytometric analysis of beads for fluorescein-derived fluorescence intensity before (grey) and after (black) immobilisation of fluorescein and DBCO-functionalised DNA (top histogram), after Esp3I treatment (2 hours at 37°C) of the DNA-coated beads (middle histogram) and after exposure of Esp3I-treated beads to a fluorescein-labelled DNA duplex that had a 5′-overhang complementary to the 5′-overhang of bead-immobilised DNA, in T4 DNA ligase buffer, with (black) or without (grey) T4 DNA ligase (bottom histogram). Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S4 . ( C ) Schematic overview of on-bead assembly allowing potential saturation of three codons in close proximity. The final, bead-attached DNA assembly is shown at the top of the panel, with the three DNA fragments used in the construction are shown below. Restriction sites are depicted in red, target codons in green and sequences used for hybridisation during ligation in blue. The first, PCR-generated amplicon (frag 3 ) was attached to bead ( via copper-free click chemistry) and digested by Esp3I. DNA on the bead was extended using an oligonucleotide duplex (frag 2 ) carrying a 5′-phosphorylated cohesive end; the sequence used to ensure stability of the duplex (stability stuffer) prior to ligation is indicated in a diagonal pattern. Once this duplex had been appended to the bead by ligation, a new cohesive end was generated (and stability stuffer removed) through BspQI digestion. Finally, another PCR amplicon (frag 1 ), separately prepared with a cohesive end (using BspQI) was ligated to the bead-immobilised DNA. Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S5 . ( D ) Flow cytometric analysis of untreated beads (top trace), beads carrying full length starting template (i.e. with FAM at one end and DBCO at the other, middle trace) and beads having gone through the 3-codon SpliMLiB process described in C. ( E ) Sanger sequencing chromatogram (templated by a PCR amplicon obtained directly from beads) of the exemplary bead-surface assembled construct shown in panel C where codons to be mutated were designed to be in close proximity (bottom). As in panel C, the green coloring refers to mutated positions, while the blue coloring refers to sequences used for ligations.
    Figure Legend Snippet: Design of bead surface and solid-phase manipulations of DNA. ( A ) Beads were designed to display both azide (labelled ‘N 3 ’) and SpyTag (labelled ‘ST’) moieties (surface modification described in Supplementary Figure S1 ). ( B ) Flow cytometric analysis of beads for fluorescein-derived fluorescence intensity before (grey) and after (black) immobilisation of fluorescein and DBCO-functionalised DNA (top histogram), after Esp3I treatment (2 hours at 37°C) of the DNA-coated beads (middle histogram) and after exposure of Esp3I-treated beads to a fluorescein-labelled DNA duplex that had a 5′-overhang complementary to the 5′-overhang of bead-immobilised DNA, in T4 DNA ligase buffer, with (black) or without (grey) T4 DNA ligase (bottom histogram). Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S4 . ( C ) Schematic overview of on-bead assembly allowing potential saturation of three codons in close proximity. The final, bead-attached DNA assembly is shown at the top of the panel, with the three DNA fragments used in the construction are shown below. Restriction sites are depicted in red, target codons in green and sequences used for hybridisation during ligation in blue. The first, PCR-generated amplicon (frag 3 ) was attached to bead ( via copper-free click chemistry) and digested by Esp3I. DNA on the bead was extended using an oligonucleotide duplex (frag 2 ) carrying a 5′-phosphorylated cohesive end; the sequence used to ensure stability of the duplex (stability stuffer) prior to ligation is indicated in a diagonal pattern. Once this duplex had been appended to the bead by ligation, a new cohesive end was generated (and stability stuffer removed) through BspQI digestion. Finally, another PCR amplicon (frag 1 ), separately prepared with a cohesive end (using BspQI) was ligated to the bead-immobilised DNA. Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S5 . ( D ) Flow cytometric analysis of untreated beads (top trace), beads carrying full length starting template (i.e. with FAM at one end and DBCO at the other, middle trace) and beads having gone through the 3-codon SpliMLiB process described in C. ( E ) Sanger sequencing chromatogram (templated by a PCR amplicon obtained directly from beads) of the exemplary bead-surface assembled construct shown in panel C where codons to be mutated were designed to be in close proximity (bottom). As in panel C, the green coloring refers to mutated positions, while the blue coloring refers to sequences used for ligations.

    Techniques Used: Modification, Derivative Assay, Fluorescence, Hybridization, Ligation, Polymerase Chain Reaction, Generated, Amplification, Sequencing, Construct

    23) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Ligation, Produced, Standard Deviation

    24) Product Images from "Split mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins"

    Article Title: Split mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa270

    Design of bead surface and solid-phase manipulations of DNA. ( A ) Beads were designed to display both azide (labelled ‘N 3 ’) and SpyTag (labelled ‘ST’) moieties (surface modification described in Supplementary Figure S1 ). ( B ) Flow cytometric analysis of beads for fluorescein-derived fluorescence intensity before (grey) and after (black) immobilisation of fluorescein and DBCO-functionalised DNA (top histogram), after Esp3I treatment (2 hours at 37°C) of the DNA-coated beads (middle histogram) and after exposure of Esp3I-treated beads to a fluorescein-labelled DNA duplex that had a 5′-overhang complementary to the 5′-overhang of bead-immobilised DNA, in T4 DNA ligase buffer, with (black) or without (grey) T4 DNA ligase (bottom histogram). Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S4 . ( C ) Schematic overview of on-bead assembly allowing potential saturation of three codons in close proximity. The final, bead-attached DNA assembly is shown at the top of the panel, with the three DNA fragments used in the construction are shown below. Restriction sites are depicted in red, target codons in green and sequences used for hybridisation during ligation in blue. The first, PCR-generated amplicon (frag 3 ) was attached to bead ( via copper-free click chemistry) and digested by Esp3I. DNA on the bead was extended using an oligonucleotide duplex (frag 2 ) carrying a 5′-phosphorylated cohesive end; the sequence used to ensure stability of the duplex (stability stuffer) prior to ligation is indicated in a diagonal pattern. Once this duplex had been appended to the bead by ligation, a new cohesive end was generated (and stability stuffer removed) through BspQI digestion. Finally, another PCR amplicon (frag 1 ), separately prepared with a cohesive end (using BspQI) was ligated to the bead-immobilised DNA. Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S5 . ( D ) Flow cytometric analysis of untreated beads (top trace), beads carrying full length starting template (i.e. with FAM at one end and DBCO at the other, middle trace) and beads having gone through the 3-codon SpliMLiB process described in C. ( E ) Sanger sequencing chromatogram (templated by a PCR amplicon obtained directly from beads) of the exemplary bead-surface assembled construct shown in panel C where codons to be mutated were designed to be in close proximity (bottom). As in panel C, the green coloring refers to mutated positions, while the blue coloring refers to sequences used for ligations.
    Figure Legend Snippet: Design of bead surface and solid-phase manipulations of DNA. ( A ) Beads were designed to display both azide (labelled ‘N 3 ’) and SpyTag (labelled ‘ST’) moieties (surface modification described in Supplementary Figure S1 ). ( B ) Flow cytometric analysis of beads for fluorescein-derived fluorescence intensity before (grey) and after (black) immobilisation of fluorescein and DBCO-functionalised DNA (top histogram), after Esp3I treatment (2 hours at 37°C) of the DNA-coated beads (middle histogram) and after exposure of Esp3I-treated beads to a fluorescein-labelled DNA duplex that had a 5′-overhang complementary to the 5′-overhang of bead-immobilised DNA, in T4 DNA ligase buffer, with (black) or without (grey) T4 DNA ligase (bottom histogram). Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S4 . ( C ) Schematic overview of on-bead assembly allowing potential saturation of three codons in close proximity. The final, bead-attached DNA assembly is shown at the top of the panel, with the three DNA fragments used in the construction are shown below. Restriction sites are depicted in red, target codons in green and sequences used for hybridisation during ligation in blue. The first, PCR-generated amplicon (frag 3 ) was attached to bead ( via copper-free click chemistry) and digested by Esp3I. DNA on the bead was extended using an oligonucleotide duplex (frag 2 ) carrying a 5′-phosphorylated cohesive end; the sequence used to ensure stability of the duplex (stability stuffer) prior to ligation is indicated in a diagonal pattern. Once this duplex had been appended to the bead by ligation, a new cohesive end was generated (and stability stuffer removed) through BspQI digestion. Finally, another PCR amplicon (frag 1 ), separately prepared with a cohesive end (using BspQI) was ligated to the bead-immobilised DNA. Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S5 . ( D ) Flow cytometric analysis of untreated beads (top trace), beads carrying full length starting template (i.e. with FAM at one end and DBCO at the other, middle trace) and beads having gone through the 3-codon SpliMLiB process described in C. ( E ) Sanger sequencing chromatogram (templated by a PCR amplicon obtained directly from beads) of the exemplary bead-surface assembled construct shown in panel C where codons to be mutated were designed to be in close proximity (bottom). As in panel C, the green coloring refers to mutated positions, while the blue coloring refers to sequences used for ligations.

    Techniques Used: Modification, Derivative Assay, Fluorescence, Hybridization, Ligation, Polymerase Chain Reaction, Generated, Amplification, Sequencing, Construct

    25) Product Images from "Biochemical Properties of a Decoy Oligodeoxynucleotide Inhibitor of STAT3 Transcription Factor"

    Article Title: Biochemical Properties of a Decoy Oligodeoxynucleotide Inhibitor of STAT3 Transcription Factor

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19061608

    Ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D) is unaffected by biotinylation. ( A ) Structures of parental and biotinylated STAT3 decoy (S3D) and CS3D. ( B ) CS3D and biotinylated CS3D were incubated with T4 DNA ligase overnight, followed by electrophoresis on a urea/polyacrylamide gel and staining with SYBR Gold.
    Figure Legend Snippet: Ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D) is unaffected by biotinylation. ( A ) Structures of parental and biotinylated STAT3 decoy (S3D) and CS3D. ( B ) CS3D and biotinylated CS3D were incubated with T4 DNA ligase overnight, followed by electrophoresis on a urea/polyacrylamide gel and staining with SYBR Gold.

    Techniques Used: Ligation, Incubation, Electrophoresis, Staining

    Efficient ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D). ( A ) Schematic representation of CS3D ligation with T4 DNA ligase. The complementary segments of the single-stranded decoy molecule spontaneously self-anneal. Enzymatic ligation with T4 DNA ligase was used to complete cyclization. ( B ) Incubations were performed in the absence or presence of T4 DNA ligase overnight. Multiple identical ligations ( n = 5) were simultaneously performed. Samples from each reaction were then electrophoresed on a urea/polyacrylamide gel, stained with SYBR Gold, and quantified by densitometry.
    Figure Legend Snippet: Efficient ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D). ( A ) Schematic representation of CS3D ligation with T4 DNA ligase. The complementary segments of the single-stranded decoy molecule spontaneously self-anneal. Enzymatic ligation with T4 DNA ligase was used to complete cyclization. ( B ) Incubations were performed in the absence or presence of T4 DNA ligase overnight. Multiple identical ligations ( n = 5) were simultaneously performed. Samples from each reaction were then electrophoresed on a urea/polyacrylamide gel, stained with SYBR Gold, and quantified by densitometry.

    Techniques Used: Ligation, Staining

    26) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Ligation, Produced, Standard Deviation

    27) Product Images from "Methylase-assisted subcloning for high throughput BioBrick assembly"

    Article Title: Methylase-assisted subcloning for high throughput BioBrick assembly

    Journal: PeerJ

    doi: 10.7717/peerj.9841

    4R/2M (PstI) BioBrick assembly. The EcoRI site of the recipient plasmid (1) and SpeI site of the insert (2) are methylated in vivo. The recipient plasmid (1) is digested with SpeI and PstI, so that it releases a short 18 bp stuffer (or “snippet”, 4). The donor plasmid (2) is separately digested with XbaI and PstI, producing the desired insert (5) and the undesired donor plasmid fragment (6). The restriction enzymes are heat-killed, the digestion products are mixed and reacted with T4 DNA ligase, forming three sets of ligation products: parental-plasmids (1-2), homodimers (7-9) and heterodimers (10-13). The 36 bp snippet homodimer is not shown, nor are trimer, tetramer and other higher order products. The homodimer products (7-9) are large perfect inverted repeats, which are not expected to replicate efficiently in vivo. Moreover, none of the undesired parental (1-2), homodimer (7-9) or heterodimers (10-11) are resistant to subsequent digestion with EcoRI and SpeI. Only the desired insert/recipient recombinant plasmid (13) retains its ability to transform E. coli .
    Figure Legend Snippet: 4R/2M (PstI) BioBrick assembly. The EcoRI site of the recipient plasmid (1) and SpeI site of the insert (2) are methylated in vivo. The recipient plasmid (1) is digested with SpeI and PstI, so that it releases a short 18 bp stuffer (or “snippet”, 4). The donor plasmid (2) is separately digested with XbaI and PstI, producing the desired insert (5) and the undesired donor plasmid fragment (6). The restriction enzymes are heat-killed, the digestion products are mixed and reacted with T4 DNA ligase, forming three sets of ligation products: parental-plasmids (1-2), homodimers (7-9) and heterodimers (10-13). The 36 bp snippet homodimer is not shown, nor are trimer, tetramer and other higher order products. The homodimer products (7-9) are large perfect inverted repeats, which are not expected to replicate efficiently in vivo. Moreover, none of the undesired parental (1-2), homodimer (7-9) or heterodimers (10-11) are resistant to subsequent digestion with EcoRI and SpeI. Only the desired insert/recipient recombinant plasmid (13) retains its ability to transform E. coli .

    Techniques Used: Plasmid Preparation, Methylation, In Vivo, Ligation, Recombinant

    2RM BioBrick assembly. The XbaI site of one cloned BioBrick part (1), and the SpeI site of another (2), are methylated in vivo. These methylated plasmids are mixed together with XbaI, SpeI and T4 DNA ligase. Each plasmid is digested by one restriction endonuclease and protected from the other. The linear digestion products (3-4) are self-ligated to form the parental plasmids (1-2), to another copy of the same molecule in one of two orientations to form a homodimer product (5-8) or to a copy of the other plasmid (again in one of two possible orientations) to form a heterodimer (9-10). The parental plasmids (1-2) and homodimers (5-8) are susceptible to re-digestion, so they are depleted over time while the heterodimers (9-10) accumulate. Both the desired (10) and undesired (9) heterodimers replicate in vivo so restriction mapping is required to differentiate between the two.
    Figure Legend Snippet: 2RM BioBrick assembly. The XbaI site of one cloned BioBrick part (1), and the SpeI site of another (2), are methylated in vivo. These methylated plasmids are mixed together with XbaI, SpeI and T4 DNA ligase. Each plasmid is digested by one restriction endonuclease and protected from the other. The linear digestion products (3-4) are self-ligated to form the parental plasmids (1-2), to another copy of the same molecule in one of two orientations to form a homodimer product (5-8) or to a copy of the other plasmid (again in one of two possible orientations) to form a heterodimer (9-10). The parental plasmids (1-2) and homodimers (5-8) are susceptible to re-digestion, so they are depleted over time while the heterodimers (9-10) accumulate. Both the desired (10) and undesired (9) heterodimers replicate in vivo so restriction mapping is required to differentiate between the two.

    Techniques Used: Clone Assay, Methylation, In Vivo, Plasmid Preparation

    28) Product Images from "Split mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins"

    Article Title: Split mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa270

    Design of bead surface and solid-phase manipulations of DNA. ( A ) Beads were designed to display both azide (labelled ‘N 3 ’) and SpyTag (labelled ‘ST’) moieties (surface modification described in Supplementary Figure S1 ). ( B ) Flow cytometric analysis of beads for fluorescein-derived fluorescence intensity before (grey) and after (black) immobilisation of fluorescein and DBCO-functionalised DNA (top histogram), after Esp3I treatment (2 hours at 37°C) of the DNA-coated beads (middle histogram) and after exposure of Esp3I-treated beads to a fluorescein-labelled DNA duplex that had a 5′-overhang complementary to the 5′-overhang of bead-immobilised DNA, in T4 DNA ligase buffer, with (black) or without (grey) T4 DNA ligase (bottom histogram). Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S4 . ( C ) Schematic overview of on-bead assembly allowing potential saturation of three codons in close proximity. The final, bead-attached DNA assembly is shown at the top of the panel, with the three DNA fragments used in the construction are shown below. Restriction sites are depicted in red, target codons in green and sequences used for hybridisation during ligation in blue. The first, PCR-generated amplicon (frag 3 ) was attached to bead ( via copper-free click chemistry) and digested by Esp3I. DNA on the bead was extended using an oligonucleotide duplex (frag 2 ) carrying a 5′-phosphorylated cohesive end; the sequence used to ensure stability of the duplex (stability stuffer) prior to ligation is indicated in a diagonal pattern. Once this duplex had been appended to the bead by ligation, a new cohesive end was generated (and stability stuffer removed) through BspQI digestion. Finally, another PCR amplicon (frag 1 ), separately prepared with a cohesive end (using BspQI) was ligated to the bead-immobilised DNA. Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S5 . ( D ) Flow cytometric analysis of untreated beads (top trace), beads carrying full length starting template (i.e. with FAM at one end and DBCO at the other, middle trace) and beads having gone through the 3-codon SpliMLiB process described in C. ( E ) Sanger sequencing chromatogram (templated by a PCR amplicon obtained directly from beads) of the exemplary bead-surface assembled construct shown in panel C where codons to be mutated were designed to be in close proximity (bottom). As in panel C, the green coloring refers to mutated positions, while the blue coloring refers to sequences used for ligations.
    Figure Legend Snippet: Design of bead surface and solid-phase manipulations of DNA. ( A ) Beads were designed to display both azide (labelled ‘N 3 ’) and SpyTag (labelled ‘ST’) moieties (surface modification described in Supplementary Figure S1 ). ( B ) Flow cytometric analysis of beads for fluorescein-derived fluorescence intensity before (grey) and after (black) immobilisation of fluorescein and DBCO-functionalised DNA (top histogram), after Esp3I treatment (2 hours at 37°C) of the DNA-coated beads (middle histogram) and after exposure of Esp3I-treated beads to a fluorescein-labelled DNA duplex that had a 5′-overhang complementary to the 5′-overhang of bead-immobilised DNA, in T4 DNA ligase buffer, with (black) or without (grey) T4 DNA ligase (bottom histogram). Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S4 . ( C ) Schematic overview of on-bead assembly allowing potential saturation of three codons in close proximity. The final, bead-attached DNA assembly is shown at the top of the panel, with the three DNA fragments used in the construction are shown below. Restriction sites are depicted in red, target codons in green and sequences used for hybridisation during ligation in blue. The first, PCR-generated amplicon (frag 3 ) was attached to bead ( via copper-free click chemistry) and digested by Esp3I. DNA on the bead was extended using an oligonucleotide duplex (frag 2 ) carrying a 5′-phosphorylated cohesive end; the sequence used to ensure stability of the duplex (stability stuffer) prior to ligation is indicated in a diagonal pattern. Once this duplex had been appended to the bead by ligation, a new cohesive end was generated (and stability stuffer removed) through BspQI digestion. Finally, another PCR amplicon (frag 1 ), separately prepared with a cohesive end (using BspQI) was ligated to the bead-immobilised DNA. Details of the DNA sequences used for the generation of this panel are set out in Supplementary Figure S5 . ( D ) Flow cytometric analysis of untreated beads (top trace), beads carrying full length starting template (i.e. with FAM at one end and DBCO at the other, middle trace) and beads having gone through the 3-codon SpliMLiB process described in C. ( E ) Sanger sequencing chromatogram (templated by a PCR amplicon obtained directly from beads) of the exemplary bead-surface assembled construct shown in panel C where codons to be mutated were designed to be in close proximity (bottom). As in panel C, the green coloring refers to mutated positions, while the blue coloring refers to sequences used for ligations.

    Techniques Used: Modification, Derivative Assay, Fluorescence, Hybridization, Ligation, Polymerase Chain Reaction, Generated, Amplification, Sequencing, Construct

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    Article Snippet: The reactions were directly loaded onto the anion-exchange HPLC followed by n -butanol extraction and lyophilization. .. RNA ligation using T4 RNA and DNA ligase Non-splinted T4 RNA based ligations were first performed on small scale reactions (typically 400 pmol RNA fragments in 10 μl reaction volume) mainly by optimizing the T4 RNA enzyme concentration (NEB) and testing the addition of BSA, whereas splinted T4 DNA based small scale ligation reactions (typically 200 pmol RNA fragments in 20 μl reaction volume) were performed by optimizing the T4 DNA enzyme concentration (NEB, fermentas or in-house ), the reaction time, the reaction temperature and testing the influence of PEG-4000. .. The DNA splints, which were added in a 1.5-fold excess compared to the RNA fragments, were annealed to the RNA fragments prior to ligation.

    Concentration Assay:

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage
    Article Snippet: The reactions were directly loaded onto the anion-exchange HPLC followed by n -butanol extraction and lyophilization. .. RNA ligation using T4 RNA and DNA ligase Non-splinted T4 RNA based ligations were first performed on small scale reactions (typically 400 pmol RNA fragments in 10 μl reaction volume) mainly by optimizing the T4 RNA enzyme concentration (NEB) and testing the addition of BSA, whereas splinted T4 DNA based small scale ligation reactions (typically 200 pmol RNA fragments in 20 μl reaction volume) were performed by optimizing the T4 DNA enzyme concentration (NEB, fermentas or in-house ), the reaction time, the reaction temperature and testing the influence of PEG-4000. .. The DNA splints, which were added in a 1.5-fold excess compared to the RNA fragments, were annealed to the RNA fragments prior to ligation.

    Article Title: YY1 Is a Structural Regulator of Enhancer-Promoter Loops
    Article Snippet: YY1: 0.25 nM DNA, 1× T4 DNA ligase buffer (NEB B0202S), H2 O 0.12 μg/μL of YY1. .. YY1 + competitor: 0.25 nM DNA, 1× T4 DNA ligase buffer (NEB B0202S), H2 O 0.12 μg/μL of YY1, 100 nM competitor DNA ( ) Assuming an extinction coefficient for YY1 of 19940 M−1 cm−1 and 75% purity, that gives an approximate YY1 molar concentration of ~3 uM. .. Reactions were incubated at 20°C for 20 minutes to allow binding of YY1 to the DNA.

    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.

    Purification:

    Article Title: Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination
    Article Snippet: After dialysis for 30 min at room temperature, 10 µL of the dialyzed mixture was transformed into electrocompetent E. coli NEB10β cells harboring pBE14 helper plasmid prepared as described previously. .. For control, 100 ng purified circular plasmid was mixed with 2 µL of T4 DNA ligase buffer in a 20 µL mixture. .. After dialysis for 30 min at room temperature, 10 µL of the dialyzed mixture was transformed into electrocompetent E. coli NEB10β cells prepared with the same method as described previously.

    Plasmid Preparation:

    Article Title: Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination
    Article Snippet: After dialysis for 30 min at room temperature, 10 µL of the dialyzed mixture was transformed into electrocompetent E. coli NEB10β cells harboring pBE14 helper plasmid prepared as described previously. .. For control, 100 ng purified circular plasmid was mixed with 2 µL of T4 DNA ligase buffer in a 20 µL mixture. .. After dialysis for 30 min at room temperature, 10 µL of the dialyzed mixture was transformed into electrocompetent E. coli NEB10β cells prepared with the same method as described previously.

    other:

    Article Title: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes
    Article Snippet: Any NEB restriction enzyme buffer may be used instead of T4 DNA ligase buffer in the Pyrite reaction so long as it is supplemented with 1 mM ATP.

    Fluorescence:

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases
    Article Snippet: Degree of ligation was not quantified; gels are used only to assign a qualitative degree of ligation under the conditions described in the text and were in general agreement between replicates. .. Ligase-DNA fluorescence anisotropy binding experimentsAnisotropy assays were performed in T4 DNA ligase reaction buffer. ..

    Binding Assay:

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases
    Article Snippet: Degree of ligation was not quantified; gels are used only to assign a qualitative degree of ligation under the conditions described in the text and were in general agreement between replicates. .. Ligase-DNA fluorescence anisotropy binding experimentsAnisotropy assays were performed in T4 DNA ligase reaction buffer. ..

    Polyacrylamide Gel Electrophoresis:

    Article Title: Assessing Protein Dynamics on Low-Complexity Single-Stranded DNA Curtains
    Article Snippet: .. PAGE-purified oligos were purchased from IDT. ssDNA circles were prepared by annealing 5 μ M phosphorylated template oligo (/5Phos/AG GAG AAA AAG AAA AAA AGA AAA GAA GG) and 4.5 μ M biotinylated primer oligo (5/Biosg/TC TCC TCC TTC T) in 1× T4 ligase reaction buffer (NEB B0202S)., Oligos were heated to 75 °C for 5 min and cooled to 4 °C at a rate of −1 °C min−1 . .. Annealed circles were ligated with the addition of 1 μ L of T4 DNA ligase (NEB M0202S) at room temperature for ~5 h. Ligated circles can be stored at 4 °C for up to 1 month.

    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.

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    New England Biolabs t4 dna ligase reaction buffer
    Assembly of low-complexity ssDNA curtains. (A) A phosphorylated template (black) and a biotinylated primer (green) are annealed and treated with <t>T4</t> DNA ligase to make minicircles. Low-complexity ssDNA composed solely of thymidine and cytidine is synthesized via rolling circle replication by phi29 DNAP. (B) Low-complexity ssDNA curtains with fluorescent end labeling. The 3′ end of the ssDNA was labeled with a fluorescent antibody. (C) RPA-GFP (green)-coated ssDNA with fluorescent end labeling (magenta). (D) Kymograph of a representative ssDNA in panel (C) with buffer flow on and off, indicating that the ssDNA is anchored to the surface via the 5′-biotin tether.
    T4 Dna Ligase Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Assembly of low-complexity ssDNA curtains. (A) A phosphorylated template (black) and a biotinylated primer (green) are annealed and treated with T4 DNA ligase to make minicircles. Low-complexity ssDNA composed solely of thymidine and cytidine is synthesized via rolling circle replication by phi29 DNAP. (B) Low-complexity ssDNA curtains with fluorescent end labeling. The 3′ end of the ssDNA was labeled with a fluorescent antibody. (C) RPA-GFP (green)-coated ssDNA with fluorescent end labeling (magenta). (D) Kymograph of a representative ssDNA in panel (C) with buffer flow on and off, indicating that the ssDNA is anchored to the surface via the 5′-biotin tether.

    Journal: Langmuir : the ACS journal of surfaces and colloids

    Article Title: Assessing Protein Dynamics on Low-Complexity Single-Stranded DNA Curtains

    doi: 10.1021/acs.langmuir.8b01812

    Figure Lengend Snippet: Assembly of low-complexity ssDNA curtains. (A) A phosphorylated template (black) and a biotinylated primer (green) are annealed and treated with T4 DNA ligase to make minicircles. Low-complexity ssDNA composed solely of thymidine and cytidine is synthesized via rolling circle replication by phi29 DNAP. (B) Low-complexity ssDNA curtains with fluorescent end labeling. The 3′ end of the ssDNA was labeled with a fluorescent antibody. (C) RPA-GFP (green)-coated ssDNA with fluorescent end labeling (magenta). (D) Kymograph of a representative ssDNA in panel (C) with buffer flow on and off, indicating that the ssDNA is anchored to the surface via the 5′-biotin tether.

    Article Snippet: PAGE-purified oligos were purchased from IDT. ssDNA circles were prepared by annealing 5 μ M phosphorylated template oligo (/5Phos/AG GAG AAA AAG AAA AAA AGA AAA GAA GG) and 4.5 μ M biotinylated primer oligo (5/Biosg/TC TCC TCC TTC T) in 1× T4 ligase reaction buffer (NEB B0202S)., Oligos were heated to 75 °C for 5 min and cooled to 4 °C at a rate of −1 °C min−1 .

    Techniques: Synthesized, End Labeling, Labeling, Recombinase Polymerase Amplification, Flow Cytometry

    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: YY1: 0.25 nM DNA, 1× T4 DNA ligase buffer (NEB B0202S), H2 O 0.12 μg/μL of YY1.

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

    Principle, reaction efficiencies and NMR evidence for isotope labeling of each stem-loop of the RsmZ RNA separately. ( a ) Sequence-specific RNase H cleavages to obtain all four isotopically labeled stem-loop fragments. The yields of the cleavage reactions before HPLC purification are indicated, the values in brackets are expressing the yield after purification. The different stem-loops are colored (SL1: magenta, SL2: green, SL3: orange, SL4: cyan). ( b ) Splinted T4 DNA ligase mediated ligations of isotope labeled (in color) and unlabeled (in black) fragments. The unlabeled fragments were obtained in a similar way as the labeled fragments. ( c ) NMR evidence for the successful segmental isotope labeling of each stem-loop separately. 1 H- 15 N-HSQC NMR spectrum of the uniformly 15 N-labeled RsmZ RNA (left) and overlay of the 1 H- 15 N-HSQC NMR spectra of the four segmentally labeled RsmZ RNAs with each stem-loop labeled separately (right). The spectra were recorded on a Bruker 600 MHz spectrometer at 10°C.

    Journal: Nucleic Acids Research

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage

    doi: 10.1093/nar/gkq756

    Figure Lengend Snippet: Principle, reaction efficiencies and NMR evidence for isotope labeling of each stem-loop of the RsmZ RNA separately. ( a ) Sequence-specific RNase H cleavages to obtain all four isotopically labeled stem-loop fragments. The yields of the cleavage reactions before HPLC purification are indicated, the values in brackets are expressing the yield after purification. The different stem-loops are colored (SL1: magenta, SL2: green, SL3: orange, SL4: cyan). ( b ) Splinted T4 DNA ligase mediated ligations of isotope labeled (in color) and unlabeled (in black) fragments. The unlabeled fragments were obtained in a similar way as the labeled fragments. ( c ) NMR evidence for the successful segmental isotope labeling of each stem-loop separately. 1 H- 15 N-HSQC NMR spectrum of the uniformly 15 N-labeled RsmZ RNA (left) and overlay of the 1 H- 15 N-HSQC NMR spectra of the four segmentally labeled RsmZ RNAs with each stem-loop labeled separately (right). The spectra were recorded on a Bruker 600 MHz spectrometer at 10°C.

    Article Snippet: RNA ligation using T4 RNA and DNA ligase Non-splinted T4 RNA based ligations were first performed on small scale reactions (typically 400 pmol RNA fragments in 10 μl reaction volume) mainly by optimizing the T4 RNA enzyme concentration (NEB) and testing the addition of BSA, whereas splinted T4 DNA based small scale ligation reactions (typically 200 pmol RNA fragments in 20 μl reaction volume) were performed by optimizing the T4 DNA enzyme concentration (NEB, fermentas or in-house ), the reaction time, the reaction temperature and testing the influence of PEG-4000.

    Techniques: Nuclear Magnetic Resonance, Labeling, Sequencing, High Performance Liquid Chromatography, Purification, Expressing

    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: Ligase-DNA fluorescence anisotropy binding experimentsAnisotropy assays were performed in T4 DNA ligase reaction buffer.

    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: Ligase-DNA fluorescence anisotropy binding experimentsAnisotropy assays were performed in T4 DNA ligase reaction buffer.

    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: Ligase-DNA fluorescence anisotropy binding experimentsAnisotropy assays were performed in T4 DNA ligase reaction buffer.

    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: Ligase-DNA fluorescence anisotropy binding experimentsAnisotropy assays were performed in T4 DNA ligase reaction buffer.

    Techniques: Ligation, Produced, Standard Deviation