Structured Review

Promega t4 dna ligase buffer
DNA transactions by recombinant AaHMGB1 proteins. (A) Preferential binding of AaHMGB1 protein to supercoiled DNA. An equimolar mixture of supercoiled and linearized plasmid pTZ19R (∼10 nM) was pre-incubated with increasing amounts of AaHMGB1 (0.5–1 µM) and the DNA–protein complexes were resolved on a 1% agarose gel, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; L, Linear DNA; Form II, relaxed circular DNA; (B) DNA supercoiling by AaHMGB1 and its truncated forms. Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I (Topo I) and AaHMGB1 recombinant proteins (7–14 µM). Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; Form II, relaxed circular DNA. (C) DNA bending by AaHMGB1 and its truncated forms. A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with recombinant proteins (25–50 nM) followed by ligation with <t>T4</t> DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. Exo III, exonuclease III. These experiments were repeated three to five times each.
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Images

1) Product Images from "The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus"

Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0040192

DNA transactions by recombinant AaHMGB1 proteins. (A) Preferential binding of AaHMGB1 protein to supercoiled DNA. An equimolar mixture of supercoiled and linearized plasmid pTZ19R (∼10 nM) was pre-incubated with increasing amounts of AaHMGB1 (0.5–1 µM) and the DNA–protein complexes were resolved on a 1% agarose gel, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; L, Linear DNA; Form II, relaxed circular DNA; (B) DNA supercoiling by AaHMGB1 and its truncated forms. Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I (Topo I) and AaHMGB1 recombinant proteins (7–14 µM). Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; Form II, relaxed circular DNA. (C) DNA bending by AaHMGB1 and its truncated forms. A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with recombinant proteins (25–50 nM) followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. Exo III, exonuclease III. These experiments were repeated three to five times each.
Figure Legend Snippet: DNA transactions by recombinant AaHMGB1 proteins. (A) Preferential binding of AaHMGB1 protein to supercoiled DNA. An equimolar mixture of supercoiled and linearized plasmid pTZ19R (∼10 nM) was pre-incubated with increasing amounts of AaHMGB1 (0.5–1 µM) and the DNA–protein complexes were resolved on a 1% agarose gel, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; L, Linear DNA; Form II, relaxed circular DNA; (B) DNA supercoiling by AaHMGB1 and its truncated forms. Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I (Topo I) and AaHMGB1 recombinant proteins (7–14 µM). Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; Form II, relaxed circular DNA. (C) DNA bending by AaHMGB1 and its truncated forms. A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with recombinant proteins (25–50 nM) followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. Exo III, exonuclease III. These experiments were repeated three to five times each.

Techniques Used: Recombinant, Binding Assay, Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Staining, Labeling, Ligation, DNA Ligation, Electrophoresis, Autoradiography

DNA bending assays by posphorylated AaHMGB1. A 32 P-labelled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of AaHMGB1 that were phosphorylated by PKA (panels A and B, lanes 5 and 2, respectively) or not (panels A and B, lanes 4 and 3, respectively), or by PKC (panels C and D, lanes 5 and 2, respectively) or not (panels C and D, lanes 4 and 3, respectively), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. These experiments were repeated five times.
Figure Legend Snippet: DNA bending assays by posphorylated AaHMGB1. A 32 P-labelled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of AaHMGB1 that were phosphorylated by PKA (panels A and B, lanes 5 and 2, respectively) or not (panels A and B, lanes 4 and 3, respectively), or by PKC (panels C and D, lanes 5 and 2, respectively) or not (panels C and D, lanes 4 and 3, respectively), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. These experiments were repeated five times.

Techniques Used: Incubation, Ligation, DNA Ligation, Electrophoresis, Autoradiography

2) Product Images from "CK2 Phosphorylation of Schistosoma mansoni HMGB1 Protein Regulates Its Cellular Traffic and Secretion but Not Its DNA Transactions"

Article Title: CK2 Phosphorylation of Schistosoma mansoni HMGB1 Protein Regulates Its Cellular Traffic and Secretion but Not Its DNA Transactions

Journal: PLoS ONE

doi: 10.1371/journal.pone.0023572

DNA supercoiling and bending assays by phosphorylated SmHMGB1. (A) Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I with 1 µg of recombinant SmHMGB1-FL or SmHMGB1-S172A/S174A that were phosphorylated (lanes 3–5) or not (lanes 6–8 and 9–11), by CK2. Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gels with ethidium bromide. Form I, supercoiled DNA; form II, relaxed circular DNA. (B) Top panel: autoradiography; bottom panel: Coomassie staining. (C) A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of recombinant proteins, that were phosphorylated (lanes 7–9) or not (lanes 4–6, 10–12, 13–15 and 16–18), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Controls are as follows: FL(c1): SmHMGB1-FL without CK2; FL(c2): SmHMGB1-FL without phosphate; FL(c3): SmHMGB1-FL without CK2 buffer. Linear: linear DNA; Lm: linear multimers. (D) Top panel: autoradiography; bottom panel: Coomassie staining. These experiments were repeated four times.
Figure Legend Snippet: DNA supercoiling and bending assays by phosphorylated SmHMGB1. (A) Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I with 1 µg of recombinant SmHMGB1-FL or SmHMGB1-S172A/S174A that were phosphorylated (lanes 3–5) or not (lanes 6–8 and 9–11), by CK2. Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gels with ethidium bromide. Form I, supercoiled DNA; form II, relaxed circular DNA. (B) Top panel: autoradiography; bottom panel: Coomassie staining. (C) A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of recombinant proteins, that were phosphorylated (lanes 7–9) or not (lanes 4–6, 10–12, 13–15 and 16–18), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Controls are as follows: FL(c1): SmHMGB1-FL without CK2; FL(c2): SmHMGB1-FL without phosphate; FL(c3): SmHMGB1-FL without CK2 buffer. Linear: linear DNA; Lm: linear multimers. (D) Top panel: autoradiography; bottom panel: Coomassie staining. These experiments were repeated four times.

Techniques Used: Plasmid Preparation, Incubation, Recombinant, Staining, Autoradiography, Labeling, Ligation, DNA Ligation, Electrophoresis

Related Articles

Clone Assay:

Article Title: A Modular Cloning Toolbox for the Generation of Chloroplast Transformation Vectors
Article Snippet: Paragraph title: Cloning of GB parts (domestication) ... Standard GB reactions were set up in 10 µl mixtures containing 75 ng of the target vector, 75 ng of the PCR products (GB parts or patches) or intermediate vectors carrying corresponding fragments, T4 DNA ligase buffer (Promega, Mannheim, Germany), 3 U of the required restriction enzyme (Bsa I or Bsm BI), and 1 U of T4 DNA ligase (Promega, Mannheim, Germany).

Article Title: Process Intensification for an Insect Antimicrobial Peptide Elastin-Like Polypeptide Fusion Produced in Redox-Engineered Escherichia coli
Article Snippet: Expression Vector The expression plasmid was created by Golden Gate (GG) cloning as previously described (Schreiber et al., ), with the addition of an 80x ELP (V48G16L16) with an N-terminal His6 tag at the fusion partner site, and a ΔI-CM intein at the cleavage site (Wood et al., , ). .. As described by Schreiber et al. , the 20-μL reaction mixture contained 40 fmol of each donor plasmid, 20 U T4 DNA ligase, 2 μL T4 DNA ligase buffer (Promega, Madison, WI, USA) and 10 U of BsaI (NEB, Ipswich, MA, USA).

Amplification:

Article Title: A Modular Cloning Toolbox for the Generation of Chloroplast Transformation Vectors
Article Snippet: The chloramphenicol resistance gene (cat ), including the appropriate promoter and terminator, was amplified in two patches from the vector pSB1C3 (Biobrick registry part, http://parts.igem.org ), while the ori and flanking regions were PCR-synthesized from pICH41306 . .. Standard GB reactions were set up in 10 µl mixtures containing 75 ng of the target vector, 75 ng of the PCR products (GB parts or patches) or intermediate vectors carrying corresponding fragments, T4 DNA ligase buffer (Promega, Mannheim, Germany), 3 U of the required restriction enzyme (Bsa I or Bsm BI), and 1 U of T4 DNA ligase (Promega, Mannheim, Germany).

Article Title: Genome-wide binding of transcription factors in inv(16) acute myeloid leukemia
Article Snippet: Adaptors were ligated to 10 μl of eluted DNA by addition of 15 μl of 2 × T4 DNA ligase buffer, 1 μl of Nextflex adaptor (see the Bio Scientific ChIP-Seq barcodes protocol for adaptor dilution; # 514120), and 4 μl T4 ligase (Promega #M180B). .. The PCR reaction was assembled in a total volume of 50 μl by adding 10 μl DNA, 2 μl Nextflex primer, 25 μl Kapa 2 × master mix (Kapa #KK2612), and 13 μl of H2 O. PCR was performed for 4 cycles using amplification conditions as mentioned in the Kapa protocol followed by purification using the Qiagen mini elute reaction clean up kit.

Article Title: Global Spread of Human Chromoblastomycosis Is Driven by Recombinant Cladophialophora carrionii and Predominantly Clonal Fonsecaea Species
Article Snippet: AFLP Fingerprinting Amplified Fragment Length Polymorphism (AFLP) analyses were performed by using 100–200 ng DNA. .. Five μl DNA was added to 15 μl restriction and ligation mixture containing 1 U of T4 DNA ligase buffer (Promega, Leiden, The Netherlands), 50 pmol HpyCH4IV adapter, 50 pmol MseI adapter, 2 U HpyCH4IV (New England Biolabs, Beverly, MA, U.S.A.), and 2 U MseI (New England Biolabs).

Synthesized:

Article Title: Engineering a Model Cell for Rational Tuning of GPCR Signaling
Article Snippet: Unless indicated, part sequences were either mutated or synthesized to remove or avoid all instances of the BsmBI, BsaI, BpiI, and NotI recognition sequences. .. Golden Gate reactions were prepared as follows: 0.1 μL of backbone vector, 0.5 μL of each plasmid, 1 μL T4 DNA ligase buffer (Promega), 0.5 μL T7 DNA Ligase (NEB), 0.5 μL restriction enzyme (BsaI or BsmBI) (NEB), and water to bring the final volume to 10 μL.

Construct:

Article Title: Engineering a Model Cell for Rational Tuning of GPCR Signaling
Article Snippet: Construction of all plasmid constructs in was achieved using Golden Gate assembly. .. Golden Gate reactions were prepared as follows: 0.1 μL of backbone vector, 0.5 μL of each plasmid, 1 μL T4 DNA ligase buffer (Promega), 0.5 μL T7 DNA Ligase (NEB), 0.5 μL restriction enzyme (BsaI or BsmBI) (NEB), and water to bring the final volume to 10 μL.

Electrophoresis:

Article Title: CK2 Phosphorylation of Schistosoma mansoni HMGB1 Protein Regulates Its Cellular Traffic and Secretion but Not Its DNA Transactions
Article Snippet: Briefly, a 32 P-labeled-66-bp or a 32 P-labeled-123-bp DNA fragments (1 nM) with cohesive BamHI ends were pre-incubated on ice for 20 min with appropriate amounts of recombinant proteins (50 ng), total (10 µg), nuclear (4 µg) or cytoplasmic (4 µg) adult worm extracts, in 1× T4 DNA ligase buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 10 mM dithiothreitol, and 0.5 mM ATP; Promega) in a final volume of 20 µl. .. Before electrophoresis, all DNA samples were deproteinized as described in the DNA supercoiling assay.

Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus
Article Snippet: Briefly, a 32 P-labeled 123-bp DNA fragment (∼1 nM) with cohesive BamHI ends were pre-incubated on ice for 20 min with appropriate amounts of recombinant proteins (25–50 nM) or total protein extracts from adult mosquitos (4 µg) in 1× T4 DNA ligase buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 10 mM dithiothreitol, and 0.5 mM ATP; Promega) in a final volume of 20 µL. .. Some of the ligation mixtures were digested after termination of ligations with ∼25 units of Exonuclease III (Promega) at 37°C for 30 min. Before electrophoresis, all DNA samples were deproteinized as described in the DNA supercoiling assay.

Incubation:

Article Title: Engineering a Model Cell for Rational Tuning of GPCR Signaling
Article Snippet: Golden Gate reactions were prepared as follows: 0.1 μL of backbone vector, 0.5 μL of each plasmid, 1 μL T4 DNA ligase buffer (Promega), 0.5 μL T7 DNA Ligase (NEB), 0.5 μL restriction enzyme (BsaI or BsmBI) (NEB), and water to bring the final volume to 10 μL. .. Reaction mixtures were then incubated in a thermocycler using the following program: (42°C for 2 min, 16°C for 5 min) x 25 cycles, followed by a final digestion step of 60°C for 10 min, and then heat inactivation at 80°C for 10 min.

Article Title: CK2 Phosphorylation of Schistosoma mansoni HMGB1 Protein Regulates Its Cellular Traffic and Secretion but Not Its DNA Transactions
Article Snippet: Briefly, a 32 P-labeled-66-bp or a 32 P-labeled-123-bp DNA fragments (1 nM) with cohesive BamHI ends were pre-incubated on ice for 20 min with appropriate amounts of recombinant proteins (50 ng), total (10 µg), nuclear (4 µg) or cytoplasmic (4 µg) adult worm extracts, in 1× T4 DNA ligase buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 10 mM dithiothreitol, and 0.5 mM ATP; Promega) in a final volume of 20 µl. .. The DNA was then ligated with T4 DNA ligase (0.6 unit/reaction; Promega) at 30°C for 30 min, and the ligation reactions were terminated by incubation of samples at 65°C for 15 min.

Article Title: Estrogen treatment induces MLL aberrations in human lymphoblastoid cells
Article Snippet: .. Single stranded linkers 11 and 25mers were annealed in the presence of T4 DNA ligase buffer (Promega, Madison, WI) by incubation in a thermocycler under the following conditions: 95°C for 5 min, 70°C for 1 sec, ramp temperature from 70°C to 25°C over 60 min, hold at 25°C for 60 min, ramp to 4°C over 60 min. .. Cells were pre-treated with zVAD.fmk (Promega, Madison, WI) for 2 h to suppress spontaneous apoptosis and apoptosis-induced cleavage of the MLL gene [ ], prior to treatment with E2/4-OH-E2.

Article Title: Engineering a Model Cell for Rational Tuning of GPCR Signaling
Article Snippet: Each oligonucleotide was then treated separately with T4 polynucleotide kinase (PNK) in the following reaction: 1 μL oligonucleotide (100 μM), 1 μL 10 x T4 DNA ligase buffer (Promega), 0.5 μL T4 PNK (NEB), and 7.5 μL H2 O. .. Reaction mixtures were then incubated in a thermocycler using the following program: (42°C for 2 min, 16°C for 5 min) x 10 cycles, followed by a final digestion step of 60°C for 10 min, and then heat inactivation at 80°C for 10 min.

Article Title: Creating a more robust 5-hydroxymethylfurfural oxidase by combining computational predictions with a novel effective library design
Article Snippet: .. The restriction–ligation reaction was set up in 20 μL volume with the components: pBAD SUMO 3.75 ng μL−1 , pUC57wt 2.5 ng μL−1 , pUC578xmutant 2.5 ng μL−1 , T4 DNA ligase buffer (Promega), T4 DNA ligase 1.5 U μL−1 (Promega), BsaI 1 U μL−1 (New England Biolabs); the thermocycler program was: incubation at 37 °C for 5 min and at 16 °C for 10 min repeated 50 times, followed by a final incubation at 50 °C for 10 min (final digestion) and at 80 °C for 10 min (enzyme inactivation). ..

Article Title: Engineering a Model Cell for Rational Tuning of GPCR Signaling
Article Snippet: The resulting fragment was then ligated into the Gpa1 C-terminal truncation vector using Golden Gate assembly, which was prepared as follows: 0.1 μL of pWS936 (50 fmol/ μL), 1 μL of the small fragment, 1 μL T4 DNA ligase buffer (Promega), 0.5 μL T7 DNA Ligase (NEB), 0.5 μL BsmBI (NEB), and water to bring the final volume to 10 μL. .. Reaction mixtures were then incubated in a thermocycler using the following program: (42°C for 2 min, 16°C for 5 min) x 10 cycles, followed by a final digestion step of 60°C for 10 min, and then heat inactivation at 80°C for 10 min.

Article Title: Genome-wide binding of transcription factors in inv(16) acute myeloid leukemia
Article Snippet: The reaction was incubated at 37 °C for 30 min followed by purification using the Qiagen mini elute reaction clean up kit. .. Adaptors were ligated to 10 μl of eluted DNA by addition of 15 μl of 2 × T4 DNA ligase buffer, 1 μl of Nextflex adaptor (see the Bio Scientific ChIP-Seq barcodes protocol for adaptor dilution; # 514120), and 4 μl T4 ligase (Promega #M180B).

Article Title: Process Intensification for an Insect Antimicrobial Peptide Elastin-Like Polypeptide Fusion Produced in Redox-Engineered Escherichia coli
Article Snippet: As described by Schreiber et al. , the 20-μL reaction mixture contained 40 fmol of each donor plasmid, 20 U T4 DNA ligase, 2 μL T4 DNA ligase buffer (Promega, Madison, WI, USA) and 10 U of BsaI (NEB, Ipswich, MA, USA). .. After warming to 37°C for 15 min, the GG mix was incubated for 50 cycles of 2 min at 37°C and 5 min at 16°C.

Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus
Article Snippet: Briefly, a 32 P-labeled 123-bp DNA fragment (∼1 nM) with cohesive BamHI ends were pre-incubated on ice for 20 min with appropriate amounts of recombinant proteins (25–50 nM) or total protein extracts from adult mosquitos (4 µg) in 1× T4 DNA ligase buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 10 mM dithiothreitol, and 0.5 mM ATP; Promega) in a final volume of 20 µL. .. The DNA was then ligated with T4 DNA ligase (0.6 unit/reaction; Promega) at 30°C for 30 min, and the ligation reactions were terminated by incubation of samples at 65°C for 15 min.

Article Title: Global Spread of Human Chromoblastomycosis Is Driven by Recombinant Cladophialophora carrionii and Predominantly Clonal Fonsecaea Species
Article Snippet: Five μl DNA was added to 15 μl restriction and ligation mixture containing 1 U of T4 DNA ligase buffer (Promega, Leiden, The Netherlands), 50 pmol HpyCH4IV adapter, 50 pmol MseI adapter, 2 U HpyCH4IV (New England Biolabs, Beverly, MA, U.S.A.), and 2 U MseI (New England Biolabs). .. The restriction ligation mixture was incubated for 1 h at 20°C and diluted five times with 10 mM Tris-HCl (pH 8.3) buffer.

Expressing:

Article Title: Engineering a Model Cell for Rational Tuning of GPCR Signaling
Article Snippet: To prepare the two plasmids for transformation, the CRISPR/Cas9 and gRNA expression plasmids were first digested with BsmBI or EcoRV, respectively, and the following size fragments were gel purified: p WS082, 1022 bp; pWS158, 10051 bp; pWS171, 10909 bp; pWS172, 10102 bp. ) using the CRISPR tool. gRNA sequences were then created using two annealed 26 bp oligonucleotides and assembled into the gRNA expression vector using a BsmbI Golden Gate assembly using the (GACT) overhang at the 5′ and (GTTT) at the 3′. .. Each oligonucleotide was then treated separately with T4 polynucleotide kinase (PNK) in the following reaction: 1 μL oligonucleotide (100 μM), 1 μL 10 x T4 DNA ligase buffer (Promega), 0.5 μL T4 PNK (NEB), and 7.5 μL H2 O.

Article Title: Engineering a Model Cell for Rational Tuning of GPCR Signaling
Article Snippet: .. The resulting fragment was then ligated into the gRNA expression vector using Golden Gate assembly, which was prepared as follows: 0.1 μL of pWS082 (50 fmol/ μL), 1 μL of the small fragment, 1 μL T4 DNA ligase buffer (Promega), 0.5 μL T7 DNA Ligase (NEB), 0.5 μL BsmBI (NEB), and water to bring the final volume to 10 μL. .. Reaction mixtures were then incubated in a thermocycler using the following program: (42°C for 2 min, 16°C for 5 min) x 10 cycles, followed by a final digestion step of 60°C for 10 min, and then heat inactivation at 80°C for 10 min.

Article Title: Process Intensification for an Insect Antimicrobial Peptide Elastin-Like Polypeptide Fusion Produced in Redox-Engineered Escherichia coli
Article Snippet: Paragraph title: Expression Vector ... As described by Schreiber et al. , the 20-μL reaction mixture contained 40 fmol of each donor plasmid, 20 U T4 DNA ligase, 2 μL T4 DNA ligase buffer (Promega, Madison, WI, USA) and 10 U of BsaI (NEB, Ipswich, MA, USA).

Transformation Assay:

Article Title: Engineering a Model Cell for Rational Tuning of GPCR Signaling
Article Snippet: To prepare the two plasmids for transformation, the CRISPR/Cas9 and gRNA expression plasmids were first digested with BsmBI or EcoRV, respectively, and the following size fragments were gel purified: p WS082, 1022 bp; pWS158, 10051 bp; pWS171, 10909 bp; pWS172, 10102 bp. ) using the CRISPR tool. gRNA sequences were then created using two annealed 26 bp oligonucleotides and assembled into the gRNA expression vector using a BsmbI Golden Gate assembly using the (GACT) overhang at the 5′ and (GTTT) at the 3′. .. Each oligonucleotide was then treated separately with T4 polynucleotide kinase (PNK) in the following reaction: 1 μL oligonucleotide (100 μM), 1 μL 10 x T4 DNA ligase buffer (Promega), 0.5 μL T4 PNK (NEB), and 7.5 μL H2 O.

Article Title: Engineering a Model Cell for Rational Tuning of GPCR Signaling
Article Snippet: The resulting fragment was then ligated into the Gpa1 C-terminal truncation vector using Golden Gate assembly, which was prepared as follows: 0.1 μL of pWS936 (50 fmol/ μL), 1 μL of the small fragment, 1 μL T4 DNA ligase buffer (Promega), 0.5 μL T7 DNA Ligase (NEB), 0.5 μL BsmBI (NEB), and water to bring the final volume to 10 μL. .. The entire reaction mixture was then transformed directly into E. coli and plated on LB medium with chloramphenicol (34 μg/mL) For a list of oligonucleotides used to create the G protein library, see .

Article Title: A Modular Cloning Toolbox for the Generation of Chloroplast Transformation Vectors
Article Snippet: Standard GB reactions were set up in 10 µl mixtures containing 75 ng of the target vector, 75 ng of the PCR products (GB parts or patches) or intermediate vectors carrying corresponding fragments, T4 DNA ligase buffer (Promega, Mannheim, Germany), 3 U of the required restriction enzyme (Bsa I or Bsm BI), and 1 U of T4 DNA ligase (Promega, Mannheim, Germany). .. One µl of each GB reaction mixture was transformed into chemically competent E. coli Top10 cells.

Electroporation Bacterial Transformation:

Article Title: Process Intensification for an Insect Antimicrobial Peptide Elastin-Like Polypeptide Fusion Produced in Redox-Engineered Escherichia coli
Article Snippet: As described by Schreiber et al. , the 20-μL reaction mixture contained 40 fmol of each donor plasmid, 20 U T4 DNA ligase, 2 μL T4 DNA ligase buffer (Promega, Madison, WI, USA) and 10 U of BsaI (NEB, Ipswich, MA, USA). .. The 2517-bp product (T7/lac+6xHis-ELP80 -TrxB-intein-IMPI(I38V)-T7) was used for bacterial transformation (for plasmid see ).

Southern Blot:

Article Title: Estrogen treatment induces MLL aberrations in human lymphoblastoid cells
Article Snippet: Paragraph title: 2.7. Ligation mediated PCR (LM-PCR) and Southern blot ... Single stranded linkers 11 and 25mers were annealed in the presence of T4 DNA ligase buffer (Promega, Madison, WI) by incubation in a thermocycler under the following conditions: 95°C for 5 min, 70°C for 1 sec, ramp temperature from 70°C to 25°C over 60 min, hold at 25°C for 60 min, ramp to 4°C over 60 min.

Ligation:

Article Title: CK2 Phosphorylation of Schistosoma mansoni HMGB1 Protein Regulates Its Cellular Traffic and Secretion but Not Its DNA Transactions
Article Snippet: Briefly, a 32 P-labeled-66-bp or a 32 P-labeled-123-bp DNA fragments (1 nM) with cohesive BamHI ends were pre-incubated on ice for 20 min with appropriate amounts of recombinant proteins (50 ng), total (10 µg), nuclear (4 µg) or cytoplasmic (4 µg) adult worm extracts, in 1× T4 DNA ligase buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 10 mM dithiothreitol, and 0.5 mM ATP; Promega) in a final volume of 20 µl. .. The DNA was then ligated with T4 DNA ligase (0.6 unit/reaction; Promega) at 30°C for 30 min, and the ligation reactions were terminated by incubation of samples at 65°C for 15 min.

Article Title: Estrogen treatment induces MLL aberrations in human lymphoblastoid cells
Article Snippet: Paragraph title: 2.7. Ligation mediated PCR (LM-PCR) and Southern blot ... Single stranded linkers 11 and 25mers were annealed in the presence of T4 DNA ligase buffer (Promega, Madison, WI) by incubation in a thermocycler under the following conditions: 95°C for 5 min, 70°C for 1 sec, ramp temperature from 70°C to 25°C over 60 min, hold at 25°C for 60 min, ramp to 4°C over 60 min.

Article Title: Genome-wide binding of transcription factors in inv(16) acute myeloid leukemia
Article Snippet: To prepare the DNA fragments for adaptor ligation an adenosine base was added to the 3′ ends of the repaired DNA by addition of 5 μl Klenow buffer, 10 μl dATP and 1 ul Klenow exo– (NEB # M0212L). .. Adaptors were ligated to 10 μl of eluted DNA by addition of 15 μl of 2 × T4 DNA ligase buffer, 1 μl of Nextflex adaptor (see the Bio Scientific ChIP-Seq barcodes protocol for adaptor dilution; # 514120), and 4 μl T4 ligase (Promega #M180B).

Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus
Article Snippet: Briefly, a 32 P-labeled 123-bp DNA fragment (∼1 nM) with cohesive BamHI ends were pre-incubated on ice for 20 min with appropriate amounts of recombinant proteins (25–50 nM) or total protein extracts from adult mosquitos (4 µg) in 1× T4 DNA ligase buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 10 mM dithiothreitol, and 0.5 mM ATP; Promega) in a final volume of 20 µL. .. The DNA was then ligated with T4 DNA ligase (0.6 unit/reaction; Promega) at 30°C for 30 min, and the ligation reactions were terminated by incubation of samples at 65°C for 15 min.

Article Title: Global Spread of Human Chromoblastomycosis Is Driven by Recombinant Cladophialophora carrionii and Predominantly Clonal Fonsecaea Species
Article Snippet: .. Five μl DNA was added to 15 μl restriction and ligation mixture containing 1 U of T4 DNA ligase buffer (Promega, Leiden, The Netherlands), 50 pmol HpyCH4IV adapter, 50 pmol MseI adapter, 2 U HpyCH4IV (New England Biolabs, Beverly, MA, U.S.A.), and 2 U MseI (New England Biolabs). .. The restriction ligation mixture was incubated for 1 h at 20°C and diluted five times with 10 mM Tris-HCl (pH 8.3) buffer.

Article Title: Genetic and Proteomic Analysis of Factors Affecting Serum Cholesterol Reduction by Lactobacillus acidophilus A4 ▿
Article Snippet: .. Chromosomal DNA was digested with TaqI and then circularized in a ligation reaction using T4 DNA ligase buffer (Promega) at a DNA concentration of 5 ng/μl. .. The ligation product was purified using a DNA purification kit (Promega) and resuspended in Tris-EDTA (TE) buffer at 10 ng/μl.

Sequencing:

Article Title: Engineering a Model Cell for Rational Tuning of GPCR Signaling
Article Snippet: An additional (TT) was included between the 5′ overhang and gRNA sequence to complete the HDV ribozyme sequence: 5′ GACTTTNNNNNNNNNNNNNNNNNNNN 3′ 3′ AANNNNNNNNNNNNNNNNNNNNCAAA 5′ Oligonucleotides were assembled into the pWS082 gRNA entry vector using the following protocol: Oligonucleotides were first resuspended at 100 μM in H2 O. .. Each oligonucleotide was then treated separately with T4 polynucleotide kinase (PNK) in the following reaction: 1 μL oligonucleotide (100 μM), 1 μL 10 x T4 DNA ligase buffer (Promega), 0.5 μL T4 PNK (NEB), and 7.5 μL H2 O.

Article Title: Creating a more robust 5-hydroxymethylfurfural oxidase by combining computational predictions with a novel effective library design
Article Snippet: The restriction–ligation reaction was set up in 20 μL volume with the components: pBAD SUMO 3.75 ng μL−1 , pUC57wt 2.5 ng μL−1 , pUC578xmutant 2.5 ng μL−1 , T4 DNA ligase buffer (Promega), T4 DNA ligase 1.5 U μL−1 (Promega), BsaI 1 U μL−1 (New England Biolabs); the thermocycler program was: incubation at 37 °C for 5 min and at 16 °C for 10 min repeated 50 times, followed by a final incubation at 50 °C for 10 min (final digestion) and at 80 °C for 10 min (enzyme inactivation). .. The hmfo variants were verified first by colony PCR (DreamTaq Green PCR Master Mix, Thermo Fisher) to determine the inserts size and then by sequencing.

Recombinant:

Article Title: CK2 Phosphorylation of Schistosoma mansoni HMGB1 Protein Regulates Its Cellular Traffic and Secretion but Not Its DNA Transactions
Article Snippet: .. Briefly, a 32 P-labeled-66-bp or a 32 P-labeled-123-bp DNA fragments (1 nM) with cohesive BamHI ends were pre-incubated on ice for 20 min with appropriate amounts of recombinant proteins (50 ng), total (10 µg), nuclear (4 µg) or cytoplasmic (4 µg) adult worm extracts, in 1× T4 DNA ligase buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 10 mM dithiothreitol, and 0.5 mM ATP; Promega) in a final volume of 20 µl. .. The DNA was then ligated with T4 DNA ligase (0.6 unit/reaction; Promega) at 30°C for 30 min, and the ligation reactions were terminated by incubation of samples at 65°C for 15 min.

Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus
Article Snippet: .. Briefly, a 32 P-labeled 123-bp DNA fragment (∼1 nM) with cohesive BamHI ends were pre-incubated on ice for 20 min with appropriate amounts of recombinant proteins (25–50 nM) or total protein extracts from adult mosquitos (4 µg) in 1× T4 DNA ligase buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 10 mM dithiothreitol, and 0.5 mM ATP; Promega) in a final volume of 20 µL. .. The DNA was then ligated with T4 DNA ligase (0.6 unit/reaction; Promega) at 30°C for 30 min, and the ligation reactions were terminated by incubation of samples at 65°C for 15 min.

ChIP-sequencing:

Article Title: Genome-wide binding of transcription factors in inv(16) acute myeloid leukemia
Article Snippet: .. Adaptors were ligated to 10 μl of eluted DNA by addition of 15 μl of 2 × T4 DNA ligase buffer, 1 μl of Nextflex adaptor (see the Bio Scientific ChIP-Seq barcodes protocol for adaptor dilution; # 514120), and 4 μl T4 ligase (Promega #M180B). .. The reaction was incubated at room temperature for 15 min. DNA was purified using the Qiagen mini elute reaction clean up kit, eluted in 10 μl of EB, and followed by PCR.

Multiplexing:

Article Title: Engineering a Model Cell for Rational Tuning of GPCR Signaling
Article Snippet: For multiplexing edits, multiple gRNA fragments can be introduced into yeast simultaneously. .. Each oligonucleotide was then treated separately with T4 polynucleotide kinase (PNK) in the following reaction: 1 μL oligonucleotide (100 μM), 1 μL 10 x T4 DNA ligase buffer (Promega), 0.5 μL T4 PNK (NEB), and 7.5 μL H2 O.

Size-exclusion Chromatography:

Article Title: Estrogen treatment induces MLL aberrations in human lymphoblastoid cells
Article Snippet: .. Single stranded linkers 11 and 25mers were annealed in the presence of T4 DNA ligase buffer (Promega, Madison, WI) by incubation in a thermocycler under the following conditions: 95°C for 5 min, 70°C for 1 sec, ramp temperature from 70°C to 25°C over 60 min, hold at 25°C for 60 min, ramp to 4°C over 60 min. .. Cells were pre-treated with zVAD.fmk (Promega, Madison, WI) for 2 h to suppress spontaneous apoptosis and apoptosis-induced cleavage of the MLL gene [ ], prior to treatment with E2/4-OH-E2.

Purification:

Article Title: Engineering a Model Cell for Rational Tuning of GPCR Signaling
Article Snippet: To prepare the two plasmids for transformation, the CRISPR/Cas9 and gRNA expression plasmids were first digested with BsmBI or EcoRV, respectively, and the following size fragments were gel purified: p WS082, 1022 bp; pWS158, 10051 bp; pWS171, 10909 bp; pWS172, 10102 bp. ) using the CRISPR tool. gRNA sequences were then created using two annealed 26 bp oligonucleotides and assembled into the gRNA expression vector using a BsmbI Golden Gate assembly using the (GACT) overhang at the 5′ and (GTTT) at the 3′. .. Each oligonucleotide was then treated separately with T4 polynucleotide kinase (PNK) in the following reaction: 1 μL oligonucleotide (100 μM), 1 μL 10 x T4 DNA ligase buffer (Promega), 0.5 μL T4 PNK (NEB), and 7.5 μL H2 O.

Article Title: A Modular Cloning Toolbox for the Generation of Chloroplast Transformation Vectors
Article Snippet: PCR products used in the GoldenBraid reactions were purified by QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). .. Standard GB reactions were set up in 10 µl mixtures containing 75 ng of the target vector, 75 ng of the PCR products (GB parts or patches) or intermediate vectors carrying corresponding fragments, T4 DNA ligase buffer (Promega, Mannheim, Germany), 3 U of the required restriction enzyme (Bsa I or Bsm BI), and 1 U of T4 DNA ligase (Promega, Mannheim, Germany).

Article Title: Genome-wide binding of transcription factors in inv(16) acute myeloid leukemia
Article Snippet: The reaction was incubated at 37 °C for 30 min followed by purification using the Qiagen mini elute reaction clean up kit. .. Adaptors were ligated to 10 μl of eluted DNA by addition of 15 μl of 2 × T4 DNA ligase buffer, 1 μl of Nextflex adaptor (see the Bio Scientific ChIP-Seq barcodes protocol for adaptor dilution; # 514120), and 4 μl T4 ligase (Promega #M180B).

Article Title: Genetic and Proteomic Analysis of Factors Affecting Serum Cholesterol Reduction by Lactobacillus acidophilus A4 ▿
Article Snippet: Chromosomal DNA was digested with TaqI and then circularized in a ligation reaction using T4 DNA ligase buffer (Promega) at a DNA concentration of 5 ng/μl. .. The ligation product was purified using a DNA purification kit (Promega) and resuspended in Tris-EDTA (TE) buffer at 10 ng/μl.

Article Title: Cell-Free Translation of Integral Membrane Proteins into Unilamelar Liposomes
Article Snippet: .. 10 × T4 DNA ligase buffer (Promega) High concentration T4 DNA ligase (Promega) pEU vector variant from Flexi Vector Digestion Reaction, step 2 Purified PCR product in PCR Cleanup plate from PCR Product Digestion Reaction Cleanup, step 6 .. Create a Ligation Master Mix containing 225 μ L of sterile, deionized water, 110 μ L of 10 × T4 ligase buffer, and 50 μ L of high-concentration T4 DNA ligase.

Polymerase Chain Reaction:

Article Title: Estrogen treatment induces MLL aberrations in human lymphoblastoid cells
Article Snippet: Paragraph title: 2.7. Ligation mediated PCR (LM-PCR) and Southern blot ... Single stranded linkers 11 and 25mers were annealed in the presence of T4 DNA ligase buffer (Promega, Madison, WI) by incubation in a thermocycler under the following conditions: 95°C for 5 min, 70°C for 1 sec, ramp temperature from 70°C to 25°C over 60 min, hold at 25°C for 60 min, ramp to 4°C over 60 min.

Article Title: Creating a more robust 5-hydroxymethylfurfural oxidase by combining computational predictions with a novel effective library design
Article Snippet: The restriction–ligation reaction was set up in 20 μL volume with the components: pBAD SUMO 3.75 ng μL−1 , pUC57wt 2.5 ng μL−1 , pUC578xmutant 2.5 ng μL−1 , T4 DNA ligase buffer (Promega), T4 DNA ligase 1.5 U μL−1 (Promega), BsaI 1 U μL−1 (New England Biolabs); the thermocycler program was: incubation at 37 °C for 5 min and at 16 °C for 10 min repeated 50 times, followed by a final incubation at 50 °C for 10 min (final digestion) and at 80 °C for 10 min (enzyme inactivation). .. The hmfo variants were verified first by colony PCR (DreamTaq Green PCR Master Mix, Thermo Fisher) to determine the inserts size and then by sequencing.

Article Title: A Modular Cloning Toolbox for the Generation of Chloroplast Transformation Vectors
Article Snippet: .. Standard GB reactions were set up in 10 µl mixtures containing 75 ng of the target vector, 75 ng of the PCR products (GB parts or patches) or intermediate vectors carrying corresponding fragments, T4 DNA ligase buffer (Promega, Mannheim, Germany), 3 U of the required restriction enzyme (Bsa I or Bsm BI), and 1 U of T4 DNA ligase (Promega, Mannheim, Germany). ..

Article Title: Genome-wide binding of transcription factors in inv(16) acute myeloid leukemia
Article Snippet: End repair was performed using 40 μl of ChIP DNA, 5 μl T4 DNA ligase buffer with 10 mM ATP, 2 μl dNTP mix (10 mM each), 1 μl of T4 DNA polymerase (NEB # M0203L), 1 μl diluted Klenow DNA polymerase (1:5, NEB # M0210L), and T4 PNK (NEB # M0201L) in a total volume of 50 μl and incubation at 20 °C for 30 min, followed by purification using the QIAquick PCR purification kit and elution in 34 μl of EB. .. Adaptors were ligated to 10 μl of eluted DNA by addition of 15 μl of 2 × T4 DNA ligase buffer, 1 μl of Nextflex adaptor (see the Bio Scientific ChIP-Seq barcodes protocol for adaptor dilution; # 514120), and 4 μl T4 ligase (Promega #M180B).

Article Title: Cell-Free Translation of Integral Membrane Proteins into Unilamelar Liposomes
Article Snippet: .. 10 × T4 DNA ligase buffer (Promega) High concentration T4 DNA ligase (Promega) pEU vector variant from Flexi Vector Digestion Reaction, step 2 Purified PCR product in PCR Cleanup plate from PCR Product Digestion Reaction Cleanup, step 6 .. Create a Ligation Master Mix containing 225 μ L of sterile, deionized water, 110 μ L of 10 × T4 ligase buffer, and 50 μ L of high-concentration T4 DNA ligase.

CRISPR:

Article Title: Engineering a Model Cell for Rational Tuning of GPCR Signaling
Article Snippet: To prepare the two plasmids for transformation, the CRISPR/Cas9 and gRNA expression plasmids were first digested with BsmBI or EcoRV, respectively, and the following size fragments were gel purified: p WS082, 1022 bp; pWS158, 10051 bp; pWS171, 10909 bp; pWS172, 10102 bp. ) using the CRISPR tool. gRNA sequences were then created using two annealed 26 bp oligonucleotides and assembled into the gRNA expression vector using a BsmbI Golden Gate assembly using the (GACT) overhang at the 5′ and (GTTT) at the 3′. .. Each oligonucleotide was then treated separately with T4 polynucleotide kinase (PNK) in the following reaction: 1 μL oligonucleotide (100 μM), 1 μL 10 x T4 DNA ligase buffer (Promega), 0.5 μL T4 PNK (NEB), and 7.5 μL H2 O.

Chromatin Immunoprecipitation:

Article Title: Genome-wide binding of transcription factors in inv(16) acute myeloid leukemia
Article Snippet: End repair was performed using 40 μl of ChIP DNA, 5 μl T4 DNA ligase buffer with 10 mM ATP, 2 μl dNTP mix (10 mM each), 1 μl of T4 DNA polymerase (NEB # M0203L), 1 μl diluted Klenow DNA polymerase (1:5, NEB # M0210L), and T4 PNK (NEB # M0201L) in a total volume of 50 μl and incubation at 20 °C for 30 min, followed by purification using the QIAquick PCR purification kit and elution in 34 μl of EB. .. Adaptors were ligated to 10 μl of eluted DNA by addition of 15 μl of 2 × T4 DNA ligase buffer, 1 μl of Nextflex adaptor (see the Bio Scientific ChIP-Seq barcodes protocol for adaptor dilution; # 514120), and 4 μl T4 ligase (Promega #M180B).

Plasmid Preparation:

Article Title: Engineering a Model Cell for Rational Tuning of GPCR Signaling
Article Snippet: .. Golden Gate reactions were prepared as follows: 0.1 μL of backbone vector, 0.5 μL of each plasmid, 1 μL T4 DNA ligase buffer (Promega), 0.5 μL T7 DNA Ligase (NEB), 0.5 μL restriction enzyme (BsaI or BsmBI) (NEB), and water to bring the final volume to 10 μL. .. Reaction mixtures were then incubated in a thermocycler using the following program: (42°C for 2 min, 16°C for 5 min) x 25 cycles, followed by a final digestion step of 60°C for 10 min, and then heat inactivation at 80°C for 10 min.

Article Title: Engineering a Model Cell for Rational Tuning of GPCR Signaling
Article Snippet: An additional (TT) was included between the 5′ overhang and gRNA sequence to complete the HDV ribozyme sequence: 5′ GACTTTNNNNNNNNNNNNNNNNNNNN 3′ 3′ AANNNNNNNNNNNNNNNNNNNNCAAA 5′ Oligonucleotides were assembled into the pWS082 gRNA entry vector using the following protocol: Oligonucleotides were first resuspended at 100 μM in H2 O. .. Each oligonucleotide was then treated separately with T4 polynucleotide kinase (PNK) in the following reaction: 1 μL oligonucleotide (100 μM), 1 μL 10 x T4 DNA ligase buffer (Promega), 0.5 μL T4 PNK (NEB), and 7.5 μL H2 O.

Article Title: Engineering a Model Cell for Rational Tuning of GPCR Signaling
Article Snippet: .. The resulting fragment was then ligated into the gRNA expression vector using Golden Gate assembly, which was prepared as follows: 0.1 μL of pWS082 (50 fmol/ μL), 1 μL of the small fragment, 1 μL T4 DNA ligase buffer (Promega), 0.5 μL T7 DNA Ligase (NEB), 0.5 μL BsmBI (NEB), and water to bring the final volume to 10 μL. .. Reaction mixtures were then incubated in a thermocycler using the following program: (42°C for 2 min, 16°C for 5 min) x 10 cycles, followed by a final digestion step of 60°C for 10 min, and then heat inactivation at 80°C for 10 min.

Article Title: Creating a more robust 5-hydroxymethylfurfural oxidase by combining computational predictions with a novel effective library design
Article Snippet: A derivative of pBAD SUMO vector designed with BsaI cutting site 5′-TGGTngagacc and ggtctcnCTTG-3′) was used as receiving vector. .. The restriction–ligation reaction was set up in 20 μL volume with the components: pBAD SUMO 3.75 ng μL−1 , pUC57wt 2.5 ng μL−1 , pUC578xmutant 2.5 ng μL−1 , T4 DNA ligase buffer (Promega), T4 DNA ligase 1.5 U μL−1 (Promega), BsaI 1 U μL−1 (New England Biolabs); the thermocycler program was: incubation at 37 °C for 5 min and at 16 °C for 10 min repeated 50 times, followed by a final incubation at 50 °C for 10 min (final digestion) and at 80 °C for 10 min (enzyme inactivation).

Article Title: Engineering a Model Cell for Rational Tuning of GPCR Signaling
Article Snippet: .. The resulting fragment was then ligated into the Gpa1 C-terminal truncation vector using Golden Gate assembly, which was prepared as follows: 0.1 μL of pWS936 (50 fmol/ μL), 1 μL of the small fragment, 1 μL T4 DNA ligase buffer (Promega), 0.5 μL T7 DNA Ligase (NEB), 0.5 μL BsmBI (NEB), and water to bring the final volume to 10 μL. .. Reaction mixtures were then incubated in a thermocycler using the following program: (42°C for 2 min, 16°C for 5 min) x 10 cycles, followed by a final digestion step of 60°C for 10 min, and then heat inactivation at 80°C for 10 min.

Article Title: A Modular Cloning Toolbox for the Generation of Chloroplast Transformation Vectors
Article Snippet: .. Standard GB reactions were set up in 10 µl mixtures containing 75 ng of the target vector, 75 ng of the PCR products (GB parts or patches) or intermediate vectors carrying corresponding fragments, T4 DNA ligase buffer (Promega, Mannheim, Germany), 3 U of the required restriction enzyme (Bsa I or Bsm BI), and 1 U of T4 DNA ligase (Promega, Mannheim, Germany). ..

Article Title: Process Intensification for an Insect Antimicrobial Peptide Elastin-Like Polypeptide Fusion Produced in Redox-Engineered Escherichia coli
Article Snippet: .. As described by Schreiber et al. , the 20-μL reaction mixture contained 40 fmol of each donor plasmid, 20 U T4 DNA ligase, 2 μL T4 DNA ligase buffer (Promega, Madison, WI, USA) and 10 U of BsaI (NEB, Ipswich, MA, USA). .. After warming to 37°C for 15 min, the GG mix was incubated for 50 cycles of 2 min at 37°C and 5 min at 16°C.

Article Title: Cell-Free Translation of Integral Membrane Proteins into Unilamelar Liposomes
Article Snippet: .. 10 × T4 DNA ligase buffer (Promega) High concentration T4 DNA ligase (Promega) pEU vector variant from Flexi Vector Digestion Reaction, step 2 Purified PCR product in PCR Cleanup plate from PCR Product Digestion Reaction Cleanup, step 6 .. Create a Ligation Master Mix containing 225 μ L of sterile, deionized water, 110 μ L of 10 × T4 ligase buffer, and 50 μ L of high-concentration T4 DNA ligase.

Next-Generation Sequencing:

Article Title: Genome-wide binding of transcription factors in inv(16) acute myeloid leukemia
Article Snippet: Paragraph title: Illumina high throughput sequencing ... Adaptors were ligated to 10 μl of eluted DNA by addition of 15 μl of 2 × T4 DNA ligase buffer, 1 μl of Nextflex adaptor (see the Bio Scientific ChIP-Seq barcodes protocol for adaptor dilution; # 514120), and 4 μl T4 ligase (Promega #M180B).

Concentration Assay:

Article Title: Engineering a Model Cell for Rational Tuning of GPCR Signaling
Article Snippet: To create the small DNA fragment, oligonucleotides were first resuspended at 100 μM concentration in H2 O. .. The resulting fragment was then ligated into the Gpa1 C-terminal truncation vector using Golden Gate assembly, which was prepared as follows: 0.1 μL of pWS936 (50 fmol/ μL), 1 μL of the small fragment, 1 μL T4 DNA ligase buffer (Promega), 0.5 μL T7 DNA Ligase (NEB), 0.5 μL BsmBI (NEB), and water to bring the final volume to 10 μL.

Article Title: Genetic and Proteomic Analysis of Factors Affecting Serum Cholesterol Reduction by Lactobacillus acidophilus A4 ▿
Article Snippet: .. Chromosomal DNA was digested with TaqI and then circularized in a ligation reaction using T4 DNA ligase buffer (Promega) at a DNA concentration of 5 ng/μl. .. The ligation product was purified using a DNA purification kit (Promega) and resuspended in Tris-EDTA (TE) buffer at 10 ng/μl.

Article Title: Cell-Free Translation of Integral Membrane Proteins into Unilamelar Liposomes
Article Snippet: .. 10 × T4 DNA ligase buffer (Promega) High concentration T4 DNA ligase (Promega) pEU vector variant from Flexi Vector Digestion Reaction, step 2 Purified PCR product in PCR Cleanup plate from PCR Product Digestion Reaction Cleanup, step 6 .. Create a Ligation Master Mix containing 225 μ L of sterile, deionized water, 110 μ L of 10 × T4 ligase buffer, and 50 μ L of high-concentration T4 DNA ligase.

DNA Purification:

Article Title: Genetic and Proteomic Analysis of Factors Affecting Serum Cholesterol Reduction by Lactobacillus acidophilus A4 ▿
Article Snippet: Chromosomal DNA was digested with TaqI and then circularized in a ligation reaction using T4 DNA ligase buffer (Promega) at a DNA concentration of 5 ng/μl. .. The ligation product was purified using a DNA purification kit (Promega) and resuspended in Tris-EDTA (TE) buffer at 10 ng/μl.

Variant Assay:

Article Title: Engineering a Model Cell for Rational Tuning of GPCR Signaling
Article Snippet: Paragraph title: Creation of the Gɑ Variant Library ... The resulting fragment was then ligated into the Gpa1 C-terminal truncation vector using Golden Gate assembly, which was prepared as follows: 0.1 μL of pWS936 (50 fmol/ μL), 1 μL of the small fragment, 1 μL T4 DNA ligase buffer (Promega), 0.5 μL T7 DNA Ligase (NEB), 0.5 μL BsmBI (NEB), and water to bring the final volume to 10 μL.

Article Title: Cell-Free Translation of Integral Membrane Proteins into Unilamelar Liposomes
Article Snippet: .. 10 × T4 DNA ligase buffer (Promega) High concentration T4 DNA ligase (Promega) pEU vector variant from Flexi Vector Digestion Reaction, step 2 Purified PCR product in PCR Cleanup plate from PCR Product Digestion Reaction Cleanup, step 6 .. Create a Ligation Master Mix containing 225 μ L of sterile, deionized water, 110 μ L of 10 × T4 ligase buffer, and 50 μ L of high-concentration T4 DNA ligase.

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    Promega t4 dna ligase buffer
    DNA transactions by recombinant AaHMGB1 proteins. (A) Preferential binding of AaHMGB1 protein to supercoiled DNA. An equimolar mixture of supercoiled and linearized plasmid pTZ19R (∼10 nM) was pre-incubated with increasing amounts of AaHMGB1 (0.5–1 µM) and the DNA–protein complexes were resolved on a 1% agarose gel, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; L, Linear DNA; Form II, relaxed circular DNA; (B) DNA supercoiling by AaHMGB1 and its truncated forms. Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I (Topo I) and AaHMGB1 recombinant proteins (7–14 µM). Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; Form II, relaxed circular DNA. (C) DNA bending by AaHMGB1 and its truncated forms. A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with recombinant proteins (25–50 nM) followed by ligation with <t>T4</t> DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. Exo III, exonuclease III. These experiments were repeated three to five times each.
    T4 Dna Ligase Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase buffer/product/Promega
    Average 94 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase buffer - by Bioz Stars, 2020-01
    94/100 stars
      Buy from Supplier

    80
    Promega concentrated t4 ligase buffer
    DNA transactions by recombinant AaHMGB1 proteins. (A) Preferential binding of AaHMGB1 protein to supercoiled DNA. An equimolar mixture of supercoiled and linearized plasmid pTZ19R (∼10 nM) was pre-incubated with increasing amounts of AaHMGB1 (0.5–1 µM) and the DNA–protein complexes were resolved on a 1% agarose gel, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; L, Linear DNA; Form II, relaxed circular DNA; (B) DNA supercoiling by AaHMGB1 and its truncated forms. Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I (Topo I) and AaHMGB1 recombinant proteins (7–14 µM). Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; Form II, relaxed circular DNA. (C) DNA bending by AaHMGB1 and its truncated forms. A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with recombinant proteins (25–50 nM) followed by ligation with <t>T4</t> DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. Exo III, exonuclease III. These experiments were repeated three to five times each.
    Concentrated T4 Ligase Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 80/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/concentrated t4 ligase buffer/product/Promega
    Average 80 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    concentrated t4 ligase buffer - by Bioz Stars, 2020-01
    80/100 stars
      Buy from Supplier

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    DNA transactions by recombinant AaHMGB1 proteins. (A) Preferential binding of AaHMGB1 protein to supercoiled DNA. An equimolar mixture of supercoiled and linearized plasmid pTZ19R (∼10 nM) was pre-incubated with increasing amounts of AaHMGB1 (0.5–1 µM) and the DNA–protein complexes were resolved on a 1% agarose gel, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; L, Linear DNA; Form II, relaxed circular DNA; (B) DNA supercoiling by AaHMGB1 and its truncated forms. Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I (Topo I) and AaHMGB1 recombinant proteins (7–14 µM). Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; Form II, relaxed circular DNA. (C) DNA bending by AaHMGB1 and its truncated forms. A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with recombinant proteins (25–50 nM) followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. Exo III, exonuclease III. These experiments were repeated three to five times each.

    Journal: PLoS ONE

    Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus

    doi: 10.1371/journal.pone.0040192

    Figure Lengend Snippet: DNA transactions by recombinant AaHMGB1 proteins. (A) Preferential binding of AaHMGB1 protein to supercoiled DNA. An equimolar mixture of supercoiled and linearized plasmid pTZ19R (∼10 nM) was pre-incubated with increasing amounts of AaHMGB1 (0.5–1 µM) and the DNA–protein complexes were resolved on a 1% agarose gel, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; L, Linear DNA; Form II, relaxed circular DNA; (B) DNA supercoiling by AaHMGB1 and its truncated forms. Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I (Topo I) and AaHMGB1 recombinant proteins (7–14 µM). Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; Form II, relaxed circular DNA. (C) DNA bending by AaHMGB1 and its truncated forms. A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with recombinant proteins (25–50 nM) followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. Exo III, exonuclease III. These experiments were repeated three to five times each.

    Article Snippet: Briefly, a 32 P-labeled 123-bp DNA fragment (∼1 nM) with cohesive BamHI ends were pre-incubated on ice for 20 min with appropriate amounts of recombinant proteins (25–50 nM) or total protein extracts from adult mosquitos (4 µg) in 1× T4 DNA ligase buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 10 mM dithiothreitol, and 0.5 mM ATP; Promega) in a final volume of 20 µL.

    Techniques: Recombinant, Binding Assay, Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Staining, Labeling, Ligation, DNA Ligation, Electrophoresis, Autoradiography

    DNA bending assays by posphorylated AaHMGB1. A 32 P-labelled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of AaHMGB1 that were phosphorylated by PKA (panels A and B, lanes 5 and 2, respectively) or not (panels A and B, lanes 4 and 3, respectively), or by PKC (panels C and D, lanes 5 and 2, respectively) or not (panels C and D, lanes 4 and 3, respectively), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. These experiments were repeated five times.

    Journal: PLoS ONE

    Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus

    doi: 10.1371/journal.pone.0040192

    Figure Lengend Snippet: DNA bending assays by posphorylated AaHMGB1. A 32 P-labelled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of AaHMGB1 that were phosphorylated by PKA (panels A and B, lanes 5 and 2, respectively) or not (panels A and B, lanes 4 and 3, respectively), or by PKC (panels C and D, lanes 5 and 2, respectively) or not (panels C and D, lanes 4 and 3, respectively), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. These experiments were repeated five times.

    Article Snippet: Briefly, a 32 P-labeled 123-bp DNA fragment (∼1 nM) with cohesive BamHI ends were pre-incubated on ice for 20 min with appropriate amounts of recombinant proteins (25–50 nM) or total protein extracts from adult mosquitos (4 µg) in 1× T4 DNA ligase buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 10 mM dithiothreitol, and 0.5 mM ATP; Promega) in a final volume of 20 µL.

    Techniques: Incubation, Ligation, DNA Ligation, Electrophoresis, Autoradiography

    DNA supercoiling and bending assays by phosphorylated SmHMGB1. (A) Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I with 1 µg of recombinant SmHMGB1-FL or SmHMGB1-S172A/S174A that were phosphorylated (lanes 3–5) or not (lanes 6–8 and 9–11), by CK2. Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gels with ethidium bromide. Form I, supercoiled DNA; form II, relaxed circular DNA. (B) Top panel: autoradiography; bottom panel: Coomassie staining. (C) A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of recombinant proteins, that were phosphorylated (lanes 7–9) or not (lanes 4–6, 10–12, 13–15 and 16–18), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Controls are as follows: FL(c1): SmHMGB1-FL without CK2; FL(c2): SmHMGB1-FL without phosphate; FL(c3): SmHMGB1-FL without CK2 buffer. Linear: linear DNA; Lm: linear multimers. (D) Top panel: autoradiography; bottom panel: Coomassie staining. These experiments were repeated four times.

    Journal: PLoS ONE

    Article Title: CK2 Phosphorylation of Schistosoma mansoni HMGB1 Protein Regulates Its Cellular Traffic and Secretion but Not Its DNA Transactions

    doi: 10.1371/journal.pone.0023572

    Figure Lengend Snippet: DNA supercoiling and bending assays by phosphorylated SmHMGB1. (A) Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I with 1 µg of recombinant SmHMGB1-FL or SmHMGB1-S172A/S174A that were phosphorylated (lanes 3–5) or not (lanes 6–8 and 9–11), by CK2. Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gels with ethidium bromide. Form I, supercoiled DNA; form II, relaxed circular DNA. (B) Top panel: autoradiography; bottom panel: Coomassie staining. (C) A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of recombinant proteins, that were phosphorylated (lanes 7–9) or not (lanes 4–6, 10–12, 13–15 and 16–18), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Controls are as follows: FL(c1): SmHMGB1-FL without CK2; FL(c2): SmHMGB1-FL without phosphate; FL(c3): SmHMGB1-FL without CK2 buffer. Linear: linear DNA; Lm: linear multimers. (D) Top panel: autoradiography; bottom panel: Coomassie staining. These experiments were repeated four times.

    Article Snippet: Briefly, a 32 P-labeled-66-bp or a 32 P-labeled-123-bp DNA fragments (1 nM) with cohesive BamHI ends were pre-incubated on ice for 20 min with appropriate amounts of recombinant proteins (50 ng), total (10 µg), nuclear (4 µg) or cytoplasmic (4 µg) adult worm extracts, in 1× T4 DNA ligase buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 10 mM dithiothreitol, and 0.5 mM ATP; Promega) in a final volume of 20 µl.

    Techniques: Plasmid Preparation, Incubation, Recombinant, Staining, Autoradiography, Labeling, Ligation, DNA Ligation, Electrophoresis