Structured Review

Roche t4 dna ligase
Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of <t>T4</t> DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.
T4 Dna Ligase, supplied by Roche, used in various techniques. Bioz Stars score: 97/100, based on 278 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism"

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism

Journal: BMC Microbiology

doi: 10.1186/1471-2180-4-21

Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.
Figure Legend Snippet: Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.

Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Amplification, Size-exclusion Chromatography, Agarose Gel Electrophoresis, Purification, Incubation, Ligation, Clone Assay

2) Product Images from "Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction"

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction

Journal: PLoS ONE

doi: 10.1371/journal.pone.0111538

Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).
Figure Legend Snippet: Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).

Techniques Used: Clone Assay, Plasmid Preparation, Sequencing, Amplification, Polymerase Chain Reaction, Ligation

3) Product Images from "Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction"

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction

Journal: PLoS ONE

doi: 10.1371/journal.pone.0111538

Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).
Figure Legend Snippet: Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).

Techniques Used: Clone Assay, Plasmid Preparation, Sequencing, Amplification, Polymerase Chain Reaction, Ligation

Related Articles

Clone Assay:

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction
Article Snippet: .. Then, 10 µl ligation reactions were set-up in multiple tubes, each containing 1 µl of 10X ligation buffer, 100 ng BsaI-digested vector, 25–100 ng T4 DNA polymerase-treated purified inserts (2–8 fold molar excess) and 1.0 unit of T4 DNA ligase (Roche) and incubated for 16 h at 16°C, 60 min at 37°C and heat inactivated at 65°C for 10 min. For electroporation, the contents of ligation reaction were pooled and 4 µl ligation mixture was directly electroporated into 100 µl of competent E. coli cells (efficiency 7–8×109 /µg) and several such electroporations were performed to obtain a library of ∼108 clones. .. Concept of the cloning strategy The cloning strategy described here is based on a combination of techniques involving the use of the type IIs restriction endonuclease BsaI to create a linearized vector with 4 base-long 5′-overhangs, and T4 DNA polymerase treatment of the insert in the presence of a single dNTP to create vector-compatible 4 base-long overhangs.

Article Title: REV7 counteracts DNA double-strand break resection and impacts PARP inhibition
Article Snippet: The PCR product and pEGFP-C1 vector were digested using EcoRI and KpnI and ligated using the T4 DNA Ligase (Roche) to generate pEGFP-h Rev7 and pEGFP-m Rev7R. .. Using pEGFP-m Rev7R as a template, pEGFP-C1 based Rev7 truncated constructs was amplified by PCR using the following primers: Forward primer: 5′-ATAT-GAATTC-G-ATGACCACCCTCACGCGC-3′ Reverse primers: mRev7R (1-55aa): 5′-ATAT-GGTACC-GGACATCTGAACCGGCAC -3′ mRev7R (1-81aa): 5′-ATAT-GGTACC-CTCCACATCGTTCTTCTCCAGG -3′ mRev7R (1-110aa): 5′-ATAT-GGTACC-GATGGACAGCAAGGGAGGC-3′ mRev7R (1-140aa): 5′-ATAT-GGTACC-GTTGTGATCCAGGACAGC -3′ mrev7R (1-183aa): 5′-ATAT-GGTACC-GTCGTGCATGTGGACATCCTG-3′ pEGFP-Rev7 mutants (shRNA resistant) were ordered as gBlocks (IDT) and cloned into the pEGFP-C1 vector using EcoRI and KpnI restriction enzymes and quick ligase (NEB).

Article Title: Proline oxidase controls proline, glutamate, and glutamine cellular concentrations in a U87 glioblastoma cell line
Article Snippet: The DNA from these two clones was digested with EcoRI and BbvCI restriction enzymes (Thermo Scientific). .. The fragments of interest were subsequently ligated (T4 DNA ligase, Roche) into pGEMT-easy and the sequence of the final construct was confirmed by automated sequencing.

Article Title: Episomal HBV persistence within transcribed host nuclear chromatin compartments involves HBx
Article Snippet: Subsequently, Sanger sequencing was utilized for amplicons cloned into pGEM-T easy (Promega) via the standard T7 sequencing primer. .. Digested samples were purified, diluted and then proximity-ligated at 16 °C using T4 DNA ligase (Roche).

Article Title: Hypoxia-targeted 131I therapy of hepatocellular cancer after systemic mesenchymal stem cell-mediated sodium iodide symporter gene delivery
Article Snippet: NIS cDNA was ligated into the pGL3 expression vector using the T4 DNA Ligase (Roche, Basel, Switzerland). .. For cloning of the pcDNA-ITR-HIF-Cherry plasmid, the HIF-responsive promoter was amplified from the pGL3-HIF-LUC vector and the fluorescent protein mCherry was amplified from the pCAG-Kosak-Cherry vector (a gift from M Rosemann, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany).

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism
Article Snippet: Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. .. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage.

Article Title: Molecular Basis of Aquaporin-7 Permeability Regulation by pH
Article Snippet: .. The purified product was cloned into the corresponding restriction sites of pUG35 digested with the same restriction enzymes, behind the MET25 promoter and in frame with GFP sequence and CYC1-T terminator, using T4 DNA Ligase (Roche, Basel, Switzerland). .. Cloning was performed according to standard protocols [ ] to construct the expression plasmid, pUG35-hAQP7.

Centrifugation:

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction
Article Snippet: One microlitre of dTTP (20 mM; 1.7 mM final concentration) was added to each tube containing the Exo-SAP treated inserts, mixed well by mild vortexing followed by brief centrifugation at 4°C, and 1.5 units (0.5 µl) of T4 DNA polymerase (New England Biolabs) were added. .. The contents were mixed well, centrifuged at 4°C and then placed in a thermocycler for incubation at 15°C for 60 min followed by heat inactivation at 75°C for 20 min. Ligation was performed in a total volume of 10 µl containing 1 µl of 10X ligation buffer (New England Biolabs), 25 ng BsaI-HF-digested vector, 1 µl of T4 DNA Polymerase-treated insert (2–5 fold molar excess relative to the vector) and 1.0 unit of T4 DNA ligase (Roche) for 16 h at 16°C followed by 1 h at 37°C and heat inactivation at 65°C for 10 min. For electroporation, the ligation mixture was diluted ten-fold in water and then 1 µl of the diluted ligation sample (containing 250 pg vector equivalent) was electroporated into 25 µl of E. coli competent cells [BL21 (DE3) RIL, Agilent Technologies, CA, USA; efficiency 3–5×108 /µg].

Amplification:

Article Title: REV7 counteracts DNA double-strand break resection and impacts PARP inhibition
Article Snippet: Mouse Rev7 was amplified from mouse lung cDNA in two parts to introduce silent mutations, making it resistant to Rev7 -sh2# (m Rev7 R). .. The PCR product and pEGFP-C1 vector were digested using EcoRI and KpnI and ligated using the T4 DNA Ligase (Roche) to generate pEGFP-h Rev7 and pEGFP-m Rev7R.

Article Title: DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes
Article Snippet: After enzyme inactivation and phenol extraction, the DNA was religated in a 7-ml volume (1,000 U T4 DNA Ligase, Roche). .. Three micrograms of 4C library DNA was amplified with Expand Long template PCR System (Roche).

Article Title: Epigenetic and transcriptional dysregulation of VWA2 associated with a MYC-driven oncogenic program in colorectal cancer
Article Snippet: Paragraph title: Methylation-sensitive amplified fragment length polymorphism (MS-AFLP) ... The digested DNA was ligated to 1.25 ml each of 5 pmol/ml Not I and 50 pmol/ml Mse I adaptor using 1 unit of T4 DNA ligase (Roche) overnight at 16 °C.

Article Title: Proline oxidase controls proline, glutamate, and glutamine cellular concentrations in a U87 glioblastoma cell line
Article Snippet: The fragments of interest were subsequently ligated (T4 DNA ligase, Roche) into pGEMT-easy and the sequence of the final construct was confirmed by automated sequencing. .. The resulting DNA encoding human wild-type PO was amplified by PCR (using the following primers: 5’–cataaagcttatggctctgaggcgcgccctg-3’; 5’-gttggtaccaaggcagggcgatggaagaggttg-3’) and subcloned into HindIII and KpnI restriction sites of the pEYFP-N1 expression vector (Clontech), allowing the upstream expression of PO fused to EYFP.

Article Title: Methylation Landscape of Human Breast Cancer Cells in Response to Dietary Compound Resveratrol
Article Snippet: At 15°C, 10 ml MseI fragments, 2 ml of ATP (10 mM) and 2 ml T4-DNA ligase (10 U; Roche, Grenzach-Wyhlen, Germany) were added, and primers and DNA fragments were ligated overnight. .. Digested DNA fragments were then treated with 1 ml proteinase K (Invitrogen, Karlsruhe, Germany) for 1 h at 37°C with subsequent heat inactivation at 80°C for 10 min. For the following amplification step, 10 ml consisting of 2 ml 10 Expand Long Template buffer 1 (Boehringer, Mannheim, Germany), 1 ml dNTPs (10 mM), 1 ml Lib1 primer (50 -TAACTAGCATGC-30), 1 ml expand long template DNA polymerase mixture (Boehringer, Mannheim, Germany) and 5 ml H2O were added to 20 ml reaction volume.

Article Title: Episomal HBV persistence within transcribed host nuclear chromatin compartments involves HBx
Article Snippet: Digested samples were purified, diluted and then proximity-ligated at 16 °C using T4 DNA ligase (Roche). .. Using specific primer pairs (Primer A: 5′-cccactgtttggctttcag-3′/Primer B: 5′-gggaaagccctacgaaccac-3′, or, respectively, Primer C: 5′-gtttctcctggctcagtttac-3′/Primer D: 5′-gtttctcytggctcagtttac-3′) adjacent to the respective ligation sites, genomic sequences associated with HBV cccDNA were amplified.

Article Title: Hypoxia-targeted 131I therapy of hepatocellular cancer after systemic mesenchymal stem cell-mediated sodium iodide symporter gene delivery
Article Snippet: NIS cDNA was ligated into the pGL3 expression vector using the T4 DNA Ligase (Roche, Basel, Switzerland). .. For cloning of the pcDNA-ITR-HIF-Cherry plasmid, the HIF-responsive promoter was amplified from the pGL3-HIF-LUC vector and the fluorescent protein mCherry was amplified from the pCAG-Kosak-Cherry vector (a gift from M Rosemann, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany).

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism
Article Snippet: Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. .. PCR amplification by using the external primers (italics in ) was performed according to the protocol described in section 1.

Article Title: Molecular Basis of Aquaporin-7 Permeability Regulation by pH
Article Snippet: The amplified product was digested with Spe I and Cla I (Roche Diagnostics® ) and purified using Wizard® SV Gel and PCR Clean-Up System kit (Promega). .. The purified product was cloned into the corresponding restriction sites of pUG35 digested with the same restriction enzymes, behind the MET25 promoter and in frame with GFP sequence and CYC1-T terminator, using T4 DNA Ligase (Roche, Basel, Switzerland).

Article Title: In silico-guided engineering of Pseudomonas putida towards growth under micro-oxic conditions
Article Snippet: DNA segments were amplified from the indicated template by colony-PCR using Phire Hot Start II DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturers’ protocol (primers are listed in Additional file : Table S2). .. DNA fragments were purified from agarose gel using the KG Gel Purification Kit (Machery-Nagel GmbH and Co. Düren, Germany) and ligated into the pSEVA638 backbone (Standardized SEVA plasmid system [ , ]) using T4 DNA Ligase (Roche Applied Science, Indianapolis, IN USA).

Expressing:

Article Title: Proline oxidase controls proline, glutamate, and glutamine cellular concentrations in a U87 glioblastoma cell line
Article Snippet: Paragraph title: Expression vector ... The fragments of interest were subsequently ligated (T4 DNA ligase, Roche) into pGEMT-easy and the sequence of the final construct was confirmed by automated sequencing.

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction
Article Snippet: The contents were mixed well, centrifuged at 4°C and then placed in a thermocycler for incubation at 15°C for 60 min followed by heat inactivation at 75°C for 20 min. Ligation was performed in a total volume of 10 µl containing 1 µl of 10X ligation buffer (New England Biolabs), 25 ng BsaI-HF-digested vector, 1 µl of T4 DNA Polymerase-treated insert (2–5 fold molar excess relative to the vector) and 1.0 unit of T4 DNA ligase (Roche) for 16 h at 16°C followed by 1 h at 37°C and heat inactivation at 65°C for 10 min. For electroporation, the ligation mixture was diluted ten-fold in water and then 1 µl of the diluted ligation sample (containing 250 pg vector equivalent) was electroporated into 25 µl of E. coli competent cells [BL21 (DE3) RIL, Agilent Technologies, CA, USA; efficiency 3–5×108 /µg]. .. To check for protein expression, individual colonies were inoculated into 150 µl MDAGAmp100 medium in a 96 well microtiter plate and cultured at 37°C with shaking for 3 h, and an aliquot of each suspension was subjected to PCR.

Article Title: Hypoxia-targeted 131I therapy of hepatocellular cancer after systemic mesenchymal stem cell-mediated sodium iodide symporter gene delivery
Article Snippet: .. NIS cDNA was ligated into the pGL3 expression vector using the T4 DNA Ligase (Roche, Basel, Switzerland). .. For cloning of the pcDNA-ITR-HIF-Cherry plasmid, the HIF-responsive promoter was amplified from the pGL3-HIF-LUC vector and the fluorescent protein mCherry was amplified from the pCAG-Kosak-Cherry vector (a gift from M Rosemann, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany).

Article Title: Molecular Basis of Aquaporin-7 Permeability Regulation by pH
Article Snippet: Paragraph title: 2.2. Heterologous Expression of hAQP7 in S. cerevisiae ... The purified product was cloned into the corresponding restriction sites of pUG35 digested with the same restriction enzymes, behind the MET25 promoter and in frame with GFP sequence and CYC1-T terminator, using T4 DNA Ligase (Roche, Basel, Switzerland).

Construct:

Article Title: REV7 counteracts DNA double-strand break resection and impacts PARP inhibition
Article Snippet: Paragraph title: Constructs ... The PCR product and pEGFP-C1 vector were digested using EcoRI and KpnI and ligated using the T4 DNA Ligase (Roche) to generate pEGFP-h Rev7 and pEGFP-m Rev7R.

Article Title: Proline oxidase controls proline, glutamate, and glutamine cellular concentrations in a U87 glioblastoma cell line
Article Snippet: .. The fragments of interest were subsequently ligated (T4 DNA ligase, Roche) into pGEMT-easy and the sequence of the final construct was confirmed by automated sequencing. .. The resulting DNA encoding human wild-type PO was amplified by PCR (using the following primers: 5’–cataaagcttatggctctgaggcgcgccctg-3’; 5’-gttggtaccaaggcagggcgatggaagaggttg-3’) and subcloned into HindIII and KpnI restriction sites of the pEYFP-N1 expression vector (Clontech), allowing the upstream expression of PO fused to EYFP.

Article Title: Hypoxia-targeted 131I therapy of hepatocellular cancer after systemic mesenchymal stem cell-mediated sodium iodide symporter gene delivery
Article Snippet: Paragraph title: Plasmid constructs ... NIS cDNA was ligated into the pGL3 expression vector using the T4 DNA Ligase (Roche, Basel, Switzerland).

Article Title: Molecular Basis of Aquaporin-7 Permeability Regulation by pH
Article Snippet: The purified product was cloned into the corresponding restriction sites of pUG35 digested with the same restriction enzymes, behind the MET25 promoter and in frame with GFP sequence and CYC1-T terminator, using T4 DNA Ligase (Roche, Basel, Switzerland). .. Cloning was performed according to standard protocols [ ] to construct the expression plasmid, pUG35-hAQP7.

Real-time Polymerase Chain Reaction:

Article Title: Episomal HBV persistence within transcribed host nuclear chromatin compartments involves HBx
Article Snippet: Digestion efficiencies were quantified by qPCR using a Corbett Rotor-Gene 6000 qPCR device and Quantitect SYBR Green Master Mix (Qiagen, Hilden, Germany) with primers covering the SpeI restriction site within the cccDNA normalized to a non-digested region. .. Digested samples were purified, diluted and then proximity-ligated at 16 °C using T4 DNA ligase (Roche).

Incubation:

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction
Article Snippet: .. Then, 10 µl ligation reactions were set-up in multiple tubes, each containing 1 µl of 10X ligation buffer, 100 ng BsaI-digested vector, 25–100 ng T4 DNA polymerase-treated purified inserts (2–8 fold molar excess) and 1.0 unit of T4 DNA ligase (Roche) and incubated for 16 h at 16°C, 60 min at 37°C and heat inactivated at 65°C for 10 min. For electroporation, the contents of ligation reaction were pooled and 4 µl ligation mixture was directly electroporated into 100 µl of competent E. coli cells (efficiency 7–8×109 /µg) and several such electroporations were performed to obtain a library of ∼108 clones. .. Concept of the cloning strategy The cloning strategy described here is based on a combination of techniques involving the use of the type IIs restriction endonuclease BsaI to create a linearized vector with 4 base-long 5′-overhangs, and T4 DNA polymerase treatment of the insert in the presence of a single dNTP to create vector-compatible 4 base-long overhangs.

Article Title: DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes
Article Snippet: Samples were then treated with 500 μg Proteinase K (Ambion) and incubated overnight at 65 °C to reverse formaldehyde crosslinking. .. After enzyme inactivation and phenol extraction, the DNA was religated in a 7-ml volume (1,000 U T4 DNA Ligase, Roche).

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction
Article Snippet: .. The contents were mixed well, centrifuged at 4°C and then placed in a thermocycler for incubation at 15°C for 60 min followed by heat inactivation at 75°C for 20 min. Ligation was performed in a total volume of 10 µl containing 1 µl of 10X ligation buffer (New England Biolabs), 25 ng BsaI-HF-digested vector, 1 µl of T4 DNA Polymerase-treated insert (2–5 fold molar excess relative to the vector) and 1.0 unit of T4 DNA ligase (Roche) for 16 h at 16°C followed by 1 h at 37°C and heat inactivation at 65°C for 10 min. For electroporation, the ligation mixture was diluted ten-fold in water and then 1 µl of the diluted ligation sample (containing 250 pg vector equivalent) was electroporated into 25 µl of E. coli competent cells [BL21 (DE3) RIL, Agilent Technologies, CA, USA; efficiency 3–5×108 /µg]. .. The resulting recombinants were analyzed by colony PCR followed by sequencing using an ABI 3730 DNA sequencer (Life Technologies Corporation, USA).

Article Title: Episomal HBV persistence within transcribed host nuclear chromatin compartments involves HBx
Article Snippet: Briefly, interacting DNA segments were cross-linked using 1% formaldehyde and nuclei were isolated using a lysis buffer containing 0.4% Tergitol (NP-40 substitute) followed by incubation at 37 °C in the presence of 0.3% SDS. .. Digested samples were purified, diluted and then proximity-ligated at 16 °C using T4 DNA ligase (Roche).

Article Title: Not All H3K4 methylations are Created Equal: Mll2/COMPASS Dependency in Primordial Germ Cell Specification
Article Snippet: The digested chromatin was diluted in 1× T4 DNA ligase buffer (Roche) with addition of T4 DNA ligase (Roche) and incubated overnight at 16 °C. .. Samples were then diluted and ligated with T4 DNA ligase (Roche) to generate the 4C template.

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism
Article Snippet: .. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. .. PCR amplification by using the external primers (italics in ) was performed according to the protocol described in section 1.

Article Title: DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes
Article Snippet: .. After heat inactivation (65 °C for 30 min), the reaction was diluted to a final volume of 7 ml with ligation buffer containing 100 U T4 DNA Ligase (Roche) and incubated at 16 °C overnight. .. Samples were then treated with 500 μg Proteinase K (Ambion) and incubated overnight at 65 °C to reverse formaldehyde crosslinking.

Cell Culture:

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction
Article Snippet: The contents were mixed well, centrifuged at 4°C and then placed in a thermocycler for incubation at 15°C for 60 min followed by heat inactivation at 75°C for 20 min. Ligation was performed in a total volume of 10 µl containing 1 µl of 10X ligation buffer (New England Biolabs), 25 ng BsaI-HF-digested vector, 1 µl of T4 DNA Polymerase-treated insert (2–5 fold molar excess relative to the vector) and 1.0 unit of T4 DNA ligase (Roche) for 16 h at 16°C followed by 1 h at 37°C and heat inactivation at 65°C for 10 min. For electroporation, the ligation mixture was diluted ten-fold in water and then 1 µl of the diluted ligation sample (containing 250 pg vector equivalent) was electroporated into 25 µl of E. coli competent cells [BL21 (DE3) RIL, Agilent Technologies, CA, USA; efficiency 3–5×108 /µg]. .. To check for protein expression, individual colonies were inoculated into 150 µl MDAGAmp100 medium in a 96 well microtiter plate and cultured at 37°C with shaking for 3 h, and an aliquot of each suspension was subjected to PCR.

Mass Spectrometry:

Article Title: Epigenetic and transcriptional dysregulation of VWA2 associated with a MYC-driven oncogenic program in colorectal cancer
Article Snippet: Paragraph title: Methylation-sensitive amplified fragment length polymorphism (MS-AFLP) ... The digested DNA was ligated to 1.25 ml each of 5 pmol/ml Not I and 50 pmol/ml Mse I adaptor using 1 unit of T4 DNA ligase (Roche) overnight at 16 °C.

Article Title: Engineered factor Xa variants retain procoagulant activity independent of direct factor Xa inhibitors
Article Snippet: T4-DNA ligase was obtained from Roche. rTAP was a generous gift from Sriram Krishnaswamy (Children’s Hospital of Philadelphia). .. Conditioned media was collected for six consecutive days, centrifuged at 4000×g to remove cellular debris, filtered over an 0.45 μm polyethersulfone membrane (Merck Millipore, Billerica, MS, USA), and supplemented with 1 mM benzamidine (Sigma Aldrich) prior to storage at −20 °C.

Genome Wide:

Article Title: Methylation Landscape of Human Breast Cancer Cells in Response to Dietary Compound Resveratrol
Article Snippet: Paragraph title: Genome-wide analysis of DNA methylation by array-PRIMES (aPRIMES) ... At 15°C, 10 ml MseI fragments, 2 ml of ATP (10 mM) and 2 ml T4-DNA ligase (10 U; Roche, Grenzach-Wyhlen, Germany) were added, and primers and DNA fragments were ligated overnight.

Modification:

Article Title: Engineered factor Xa variants retain procoagulant activity independent of direct factor Xa inhibitors
Article Snippet: T4-DNA ligase was obtained from Roche. rTAP was a generous gift from Sriram Krishnaswamy (Children’s Hospital of Philadelphia). .. Recombinant FV-810 was large-scale expressed in BHK cells in triple flasks (Nalge Nunc, Rochester, NY, USA) in Dulbecco’s modified Eagle’s medium/F-12 without phenol red supplemented with 2 mM l -glutamine, 100 U ml−1 penicillin, 0.1 mg ml−1 streptomycin, 0.25 μg ml−1 amphotericin B, 100 μg ml−1 geneticin, 10 μg ml−1 ITS, and 2.5 mM CaCl2 .

Article Title: Hypoxia-targeted 131I therapy of hepatocellular cancer after systemic mesenchymal stem cell-mediated sodium iodide symporter gene delivery
Article Snippet: The expression vector pGL3-HIF-LUC (a gift from F Grässer, Universitätsklinikum des Saarlandes, Homburg, Germany) was modified to contain a synthetic promoter composed of a minimal thymidine kinase promoter with six HIF-responsive elements driving transgene expression and a CMV controlled Bsr2 blasticidin resistance gene for selection of transfected cells. .. NIS cDNA was ligated into the pGL3 expression vector using the T4 DNA Ligase (Roche, Basel, Switzerland).

Article Title: Molecular Basis of Aquaporin-7 Permeability Regulation by pH
Article Snippet: Specific primers modified to incorporate restriction sites for Spe I (bold) and Cla I (bold) were designed and used for amplification of hAQP7 cDNA ( ). .. The purified product was cloned into the corresponding restriction sites of pUG35 digested with the same restriction enzymes, behind the MET25 promoter and in frame with GFP sequence and CYC1-T terminator, using T4 DNA Ligase (Roche, Basel, Switzerland).

Hybridization:

Article Title: Methylation Landscape of Human Breast Cancer Cells in Response to Dietary Compound Resveratrol
Article Snippet: To determine the methylated and unmethylated DNA regions in the promoters of genes, we used Array-PRIMES method (aPRIMES) as described previously (12). aPRIMES is based on the differential restriction and competitive hybridization of DNA by methylation-specific and methylation-sensitive restriction enzymes, respectively. .. At 15°C, 10 ml MseI fragments, 2 ml of ATP (10 mM) and 2 ml T4-DNA ligase (10 U; Roche, Grenzach-Wyhlen, Germany) were added, and primers and DNA fragments were ligated overnight.

Electroporation:

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction
Article Snippet: .. Then, 10 µl ligation reactions were set-up in multiple tubes, each containing 1 µl of 10X ligation buffer, 100 ng BsaI-digested vector, 25–100 ng T4 DNA polymerase-treated purified inserts (2–8 fold molar excess) and 1.0 unit of T4 DNA ligase (Roche) and incubated for 16 h at 16°C, 60 min at 37°C and heat inactivated at 65°C for 10 min. For electroporation, the contents of ligation reaction were pooled and 4 µl ligation mixture was directly electroporated into 100 µl of competent E. coli cells (efficiency 7–8×109 /µg) and several such electroporations were performed to obtain a library of ∼108 clones. .. Concept of the cloning strategy The cloning strategy described here is based on a combination of techniques involving the use of the type IIs restriction endonuclease BsaI to create a linearized vector with 4 base-long 5′-overhangs, and T4 DNA polymerase treatment of the insert in the presence of a single dNTP to create vector-compatible 4 base-long overhangs.

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction
Article Snippet: .. The contents were mixed well, centrifuged at 4°C and then placed in a thermocycler for incubation at 15°C for 60 min followed by heat inactivation at 75°C for 20 min. Ligation was performed in a total volume of 10 µl containing 1 µl of 10X ligation buffer (New England Biolabs), 25 ng BsaI-HF-digested vector, 1 µl of T4 DNA Polymerase-treated insert (2–5 fold molar excess relative to the vector) and 1.0 unit of T4 DNA ligase (Roche) for 16 h at 16°C followed by 1 h at 37°C and heat inactivation at 65°C for 10 min. For electroporation, the ligation mixture was diluted ten-fold in water and then 1 µl of the diluted ligation sample (containing 250 pg vector equivalent) was electroporated into 25 µl of E. coli competent cells [BL21 (DE3) RIL, Agilent Technologies, CA, USA; efficiency 3–5×108 /µg]. .. The resulting recombinants were analyzed by colony PCR followed by sequencing using an ABI 3730 DNA sequencer (Life Technologies Corporation, USA).

Flow Cytometry:

Article Title: Engineered factor Xa variants retain procoagulant activity independent of direct factor Xa inhibitors
Article Snippet: T4-DNA ligase was obtained from Roche. rTAP was a generous gift from Sriram Krishnaswamy (Children’s Hospital of Philadelphia). .. Conditioned media (6 l) was thawed at 37 °C and applied at ambient temperatures to a 2.5 × 6 cm SP Sepharose Fast Flow column (GE Healthcare, Chicago, IL, USA) equilibrated in 20 mM Hepes, 0.15 M NaCl, 5 mM CaCl2 ,1 mM benzamidine, pH 7.4.

Inverse PCR:

Article Title: DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes
Article Snippet: After enzyme inactivation and phenol extraction, the DNA was religated in a 7-ml volume (1,000 U T4 DNA Ligase, Roche). .. Baits were amplified with inverse PCR primers as follows: Igh with HindIII: IgH_R_4C 5′-CCAGACATGTGG GCTGAGAT-3′, Igh_Hind_Read 5′-CTACCCACCTAACTCCAAGC-3′; Mycn with HindIII: Mycn_R_4C 5′-CTCCCATTTTGCACTCCTGT-3′, Mycn_Hind_Read 5′-GATTTATCCTTAAACCCTTAAGC-3′; Igh with BglII: IgH_Bgl_R_4C 5′-CATGGACATTTGCGTGTGTA-3′, IgH_Bgl_Read 5′-GTG CCCCCAGGAGCAGATCT-3′; Mycn with BglII: Mycn_Bgl_R_4C 5′-AG TCTGCGGGAGGTAAGAAG-3′, Mycn_Bgl_Read 5′-CCCTTTTAGACAGCC AGATCT-3′; Myc with BglII: Myc_Bgl_R_4C 5′-AAGAATGTGCCCAGTC AACA-3′, Myc_Bgl_Read 5′-AGTGAATTGCCAACCCAGAT-3′; Rplp2 with HindIII: 5′-GCCATCTCTCCAGTCAAAAAGC-3′, CTTCTCACTTCCATT CCCTGAG-3′; Rac2 with HindIII: 5′-GCCATGGAGACCGGAAGCTT-3′, 5′-GGGACTGTCCACTCCACCT-3′; Eμ with HindIII: 5′-TGTGGCTGCTGC TCTTAAAGC-3′, 5′-TGTGAAGCCGTTTTGACCAGAATGT-3′.

Ligation:

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction
Article Snippet: .. Then, 10 µl ligation reactions were set-up in multiple tubes, each containing 1 µl of 10X ligation buffer, 100 ng BsaI-digested vector, 25–100 ng T4 DNA polymerase-treated purified inserts (2–8 fold molar excess) and 1.0 unit of T4 DNA ligase (Roche) and incubated for 16 h at 16°C, 60 min at 37°C and heat inactivated at 65°C for 10 min. For electroporation, the contents of ligation reaction were pooled and 4 µl ligation mixture was directly electroporated into 100 µl of competent E. coli cells (efficiency 7–8×109 /µg) and several such electroporations were performed to obtain a library of ∼108 clones. .. Concept of the cloning strategy The cloning strategy described here is based on a combination of techniques involving the use of the type IIs restriction endonuclease BsaI to create a linearized vector with 4 base-long 5′-overhangs, and T4 DNA polymerase treatment of the insert in the presence of a single dNTP to create vector-compatible 4 base-long overhangs.

Article Title: DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes
Article Snippet: For circularization, the ligation junctions were digested with Csp6I (Fermentas) or DpnII (New England Biolabs) at 37 °C overnight. .. After enzyme inactivation and phenol extraction, the DNA was religated in a 7-ml volume (1,000 U T4 DNA Ligase, Roche).

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction
Article Snippet: .. The contents were mixed well, centrifuged at 4°C and then placed in a thermocycler for incubation at 15°C for 60 min followed by heat inactivation at 75°C for 20 min. Ligation was performed in a total volume of 10 µl containing 1 µl of 10X ligation buffer (New England Biolabs), 25 ng BsaI-HF-digested vector, 1 µl of T4 DNA Polymerase-treated insert (2–5 fold molar excess relative to the vector) and 1.0 unit of T4 DNA ligase (Roche) for 16 h at 16°C followed by 1 h at 37°C and heat inactivation at 65°C for 10 min. For electroporation, the ligation mixture was diluted ten-fold in water and then 1 µl of the diluted ligation sample (containing 250 pg vector equivalent) was electroporated into 25 µl of E. coli competent cells [BL21 (DE3) RIL, Agilent Technologies, CA, USA; efficiency 3–5×108 /µg]. .. The resulting recombinants were analyzed by colony PCR followed by sequencing using an ABI 3730 DNA sequencer (Life Technologies Corporation, USA).

Article Title: Episomal HBV persistence within transcribed host nuclear chromatin compartments involves HBx
Article Snippet: Digested samples were purified, diluted and then proximity-ligated at 16 °C using T4 DNA ligase (Roche). .. Subsequently, samples were subjected to digestion with HaeIII (NEB, 20 U per µL, 24 h) followed by a second ligation.

Article Title: DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes
Article Snippet: .. After heat inactivation (65 °C for 30 min), the reaction was diluted to a final volume of 7 ml with ligation buffer containing 100 U T4 DNA Ligase (Roche) and incubated at 16 °C overnight. .. Samples were then treated with 500 μg Proteinase K (Ambion) and incubated overnight at 65 °C to reverse formaldehyde crosslinking.

Genomic Sequencing:

Article Title: Episomal HBV persistence within transcribed host nuclear chromatin compartments involves HBx
Article Snippet: Digested samples were purified, diluted and then proximity-ligated at 16 °C using T4 DNA ligase (Roche). .. Using specific primer pairs (Primer A: 5′-cccactgtttggctttcag-3′/Primer B: 5′-gggaaagccctacgaaccac-3′, or, respectively, Primer C: 5′-gtttctcctggctcagtttac-3′/Primer D: 5′-gtttctcytggctcagtttac-3′) adjacent to the respective ligation sites, genomic sequences associated with HBV cccDNA were amplified.

Introduce:

Article Title: REV7 counteracts DNA double-strand break resection and impacts PARP inhibition
Article Snippet: Mouse Rev7 was amplified from mouse lung cDNA in two parts to introduce silent mutations, making it resistant to Rev7 -sh2# (m Rev7 R). .. The PCR product and pEGFP-C1 vector were digested using EcoRI and KpnI and ligated using the T4 DNA Ligase (Roche) to generate pEGFP-h Rev7 and pEGFP-m Rev7R.

Mutagenesis:

Article Title: Proline oxidase controls proline, glutamate, and glutamine cellular concentrations in a U87 glioblastoma cell line
Article Snippet: The fragments of interest were subsequently ligated (T4 DNA ligase, Roche) into pGEMT-easy and the sequence of the final construct was confirmed by automated sequencing. .. The L441P mutation was introduced into the pEYFP-N1-prodh or pcDNA3-prodh constructs by using the QuikChange Site-Directed Mutagenesis Kit (Stratagene); successful mutagenesis was confirmed by DNA sequencing.

Article Title: Engineered factor Xa variants retain procoagulant activity independent of direct factor Xa inhibitors
Article Snippet: The Q5 site-directed mutagenesis kit and restriction endonucleases Bgl2, Hind3, and Apa1 were obtained from New England Biolabs (Ipswich, MA, USA). .. T4-DNA ligase was obtained from Roche. rTAP was a generous gift from Sriram Krishnaswamy (Children’s Hospital of Philadelphia).

DNA Sequencing:

Article Title: Proline oxidase controls proline, glutamate, and glutamine cellular concentrations in a U87 glioblastoma cell line
Article Snippet: The fragments of interest were subsequently ligated (T4 DNA ligase, Roche) into pGEMT-easy and the sequence of the final construct was confirmed by automated sequencing. .. The L441P mutation was introduced into the pEYFP-N1-prodh or pcDNA3-prodh constructs by using the QuikChange Site-Directed Mutagenesis Kit (Stratagene); successful mutagenesis was confirmed by DNA sequencing.

Article Title: Molecular Basis of Aquaporin-7 Permeability Regulation by pH
Article Snippet: The purified product was cloned into the corresponding restriction sites of pUG35 digested with the same restriction enzymes, behind the MET25 promoter and in frame with GFP sequence and CYC1-T terminator, using T4 DNA Ligase (Roche, Basel, Switzerland). .. Fidelity of constructs and correct orientation was verified by PCR amplification, restriction analysis, and DNA sequencing.

Sequencing:

Article Title: REV7 counteracts DNA double-strand break resection and impacts PARP inhibition
Article Snippet: Equal parts of both PCR reactions were mixed and used for a PCR using IB11m and IB12m to create the complete mouse Rev7 sequence including the silent mutations. .. The PCR product and pEGFP-C1 vector were digested using EcoRI and KpnI and ligated using the T4 DNA Ligase (Roche) to generate pEGFP-h Rev7 and pEGFP-m Rev7R.

Article Title: Proline oxidase controls proline, glutamate, and glutamine cellular concentrations in a U87 glioblastoma cell line
Article Snippet: .. The fragments of interest were subsequently ligated (T4 DNA ligase, Roche) into pGEMT-easy and the sequence of the final construct was confirmed by automated sequencing. .. The resulting DNA encoding human wild-type PO was amplified by PCR (using the following primers: 5’–cataaagcttatggctctgaggcgcgccctg-3’; 5’-gttggtaccaaggcagggcgatggaagaggttg-3’) and subcloned into HindIII and KpnI restriction sites of the pEYFP-N1 expression vector (Clontech), allowing the upstream expression of PO fused to EYFP.

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction
Article Snippet: The contents were mixed well, centrifuged at 4°C and then placed in a thermocycler for incubation at 15°C for 60 min followed by heat inactivation at 75°C for 20 min. Ligation was performed in a total volume of 10 µl containing 1 µl of 10X ligation buffer (New England Biolabs), 25 ng BsaI-HF-digested vector, 1 µl of T4 DNA Polymerase-treated insert (2–5 fold molar excess relative to the vector) and 1.0 unit of T4 DNA ligase (Roche) for 16 h at 16°C followed by 1 h at 37°C and heat inactivation at 65°C for 10 min. For electroporation, the ligation mixture was diluted ten-fold in water and then 1 µl of the diluted ligation sample (containing 250 pg vector equivalent) was electroporated into 25 µl of E. coli competent cells [BL21 (DE3) RIL, Agilent Technologies, CA, USA; efficiency 3–5×108 /µg]. .. The resulting recombinants were analyzed by colony PCR followed by sequencing using an ABI 3730 DNA sequencer (Life Technologies Corporation, USA).

Article Title: Episomal HBV persistence within transcribed host nuclear chromatin compartments involves HBx
Article Snippet: Subsequently, Sanger sequencing was utilized for amplicons cloned into pGEM-T easy (Promega) via the standard T7 sequencing primer. .. Digested samples were purified, diluted and then proximity-ligated at 16 °C using T4 DNA ligase (Roche).

Article Title: Not All H3K4 methylations are Created Equal: Mll2/COMPASS Dependency in Primordial Germ Cell Specification
Article Snippet: Paragraph title: 4C Sequencing ... Samples were then diluted and ligated with T4 DNA ligase (Roche) to generate the 4C template.

Article Title: Molecular Basis of Aquaporin-7 Permeability Regulation by pH
Article Snippet: .. The purified product was cloned into the corresponding restriction sites of pUG35 digested with the same restriction enzymes, behind the MET25 promoter and in frame with GFP sequence and CYC1-T terminator, using T4 DNA Ligase (Roche, Basel, Switzerland). .. Cloning was performed according to standard protocols [ ] to construct the expression plasmid, pUG35-hAQP7.

Recombinant:

Article Title: Engineered factor Xa variants retain procoagulant activity independent of direct factor Xa inhibitors
Article Snippet: T4-DNA ligase was obtained from Roche. rTAP was a generous gift from Sriram Krishnaswamy (Children’s Hospital of Philadelphia). .. Recombinant FV-810 was large-scale expressed in BHK cells in triple flasks (Nalge Nunc, Rochester, NY, USA) in Dulbecco’s modified Eagle’s medium/F-12 without phenol red supplemented with 2 mM l -glutamine, 100 U ml−1 penicillin, 0.1 mg ml−1 streptomycin, 0.25 μg ml−1 amphotericin B, 100 μg ml−1 geneticin, 10 μg ml−1 ITS, and 2.5 mM CaCl2 .

Molecular Weight:

Article Title: Methylation Landscape of Human Breast Cancer Cells in Response to Dietary Compound Resveratrol
Article Snippet: Genome-wide analysis of DNA methylation by array-PRIMES (aPRIMES) The extraction of high molecular weight DNA of the cells MDA-MB-231 untreated and treated with resveratrol was extracted using the DNeasy Kit (Qiagen, Germany) according to the manufacturer’s instructions. .. At 15°C, 10 ml MseI fragments, 2 ml of ATP (10 mM) and 2 ml T4-DNA ligase (10 U; Roche, Grenzach-Wyhlen, Germany) were added, and primers and DNA fragments were ligated overnight.

Methylation:

Article Title: Epigenetic and transcriptional dysregulation of VWA2 associated with a MYC-driven oncogenic program in colorectal cancer
Article Snippet: Paragraph title: Methylation-sensitive amplified fragment length polymorphism (MS-AFLP) ... The digested DNA was ligated to 1.25 ml each of 5 pmol/ml Not I and 50 pmol/ml Mse I adaptor using 1 unit of T4 DNA ligase (Roche) overnight at 16 °C.

Article Title: Methylation Landscape of Human Breast Cancer Cells in Response to Dietary Compound Resveratrol
Article Snippet: To determine the methylated and unmethylated DNA regions in the promoters of genes, we used Array-PRIMES method (aPRIMES) as described previously (12). aPRIMES is based on the differential restriction and competitive hybridization of DNA by methylation-specific and methylation-sensitive restriction enzymes, respectively. .. At 15°C, 10 ml MseI fragments, 2 ml of ATP (10 mM) and 2 ml T4-DNA ligase (10 U; Roche, Grenzach-Wyhlen, Germany) were added, and primers and DNA fragments were ligated overnight.

Multiple Displacement Amplification:

Article Title: Methylation Landscape of Human Breast Cancer Cells in Response to Dietary Compound Resveratrol
Article Snippet: Genome-wide analysis of DNA methylation by array-PRIMES (aPRIMES) The extraction of high molecular weight DNA of the cells MDA-MB-231 untreated and treated with resveratrol was extracted using the DNeasy Kit (Qiagen, Germany) according to the manufacturer’s instructions. .. At 15°C, 10 ml MseI fragments, 2 ml of ATP (10 mM) and 2 ml T4-DNA ligase (10 U; Roche, Grenzach-Wyhlen, Germany) were added, and primers and DNA fragments were ligated overnight.

Isolation:

Article Title: Episomal HBV persistence within transcribed host nuclear chromatin compartments involves HBx
Article Snippet: Briefly, interacting DNA segments were cross-linked using 1% formaldehyde and nuclei were isolated using a lysis buffer containing 0.4% Tergitol (NP-40 substitute) followed by incubation at 37 °C in the presence of 0.3% SDS. .. Digested samples were purified, diluted and then proximity-ligated at 16 °C using T4 DNA ligase (Roche).

Article Title: Molecular Basis of Aquaporin-7 Permeability Regulation by pH
Article Snippet: Plasmidic DNA was isolated and purified. .. The purified product was cloned into the corresponding restriction sites of pUG35 digested with the same restriction enzymes, behind the MET25 promoter and in frame with GFP sequence and CYC1-T terminator, using T4 DNA Ligase (Roche, Basel, Switzerland).

Article Title: In silico-guided engineering of Pseudomonas putida towards growth under micro-oxic conditions
Article Snippet: Restriction enzymes were obtained from NEB (New England \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text {BioLabs}\circledR \text {inc}$$\end{document} BioLabs ® inc , Ipswich, MA USA.). l -aspartate oxidase was isolated from both E. coli BW25113 or P. putida KT2440. .. DNA fragments were purified from agarose gel using the KG Gel Purification Kit (Machery-Nagel GmbH and Co. Düren, Germany) and ligated into the pSEVA638 backbone (Standardized SEVA plasmid system [ , ]) using T4 DNA Ligase (Roche Applied Science, Indianapolis, IN USA).

Transfection:

Article Title: Hypoxia-targeted 131I therapy of hepatocellular cancer after systemic mesenchymal stem cell-mediated sodium iodide symporter gene delivery
Article Snippet: The expression vector pGL3-HIF-LUC (a gift from F Grässer, Universitätsklinikum des Saarlandes, Homburg, Germany) was modified to contain a synthetic promoter composed of a minimal thymidine kinase promoter with six HIF-responsive elements driving transgene expression and a CMV controlled Bsr2 blasticidin resistance gene for selection of transfected cells. .. NIS cDNA was ligated into the pGL3 expression vector using the T4 DNA Ligase (Roche, Basel, Switzerland).

Labeling:

Article Title: Epigenetic and transcriptional dysregulation of VWA2 associated with a MYC-driven oncogenic program in colorectal cancer
Article Snippet: The digested DNA was ligated to 1.25 ml each of 5 pmol/ml Not I and 50 pmol/ml Mse I adaptor using 1 unit of T4 DNA ligase (Roche) overnight at 16 °C. .. A primer complementary to the NotI adaptor (Not I primer) was labeled at the 5′ end using 32 P-γ-ATP and T4 polynucleotide kinase (Promega, Madison, WI).

Purification:

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction
Article Snippet: .. Then, 10 µl ligation reactions were set-up in multiple tubes, each containing 1 µl of 10X ligation buffer, 100 ng BsaI-digested vector, 25–100 ng T4 DNA polymerase-treated purified inserts (2–8 fold molar excess) and 1.0 unit of T4 DNA ligase (Roche) and incubated for 16 h at 16°C, 60 min at 37°C and heat inactivated at 65°C for 10 min. For electroporation, the contents of ligation reaction were pooled and 4 µl ligation mixture was directly electroporated into 100 µl of competent E. coli cells (efficiency 7–8×109 /µg) and several such electroporations were performed to obtain a library of ∼108 clones. .. Concept of the cloning strategy The cloning strategy described here is based on a combination of techniques involving the use of the type IIs restriction endonuclease BsaI to create a linearized vector with 4 base-long 5′-overhangs, and T4 DNA polymerase treatment of the insert in the presence of a single dNTP to create vector-compatible 4 base-long overhangs.

Article Title: REV7 counteracts DNA double-strand break resection and impacts PARP inhibition
Article Snippet: The PCR product and pEGFP-C1 vector were digested using EcoRI and KpnI and ligated using the T4 DNA Ligase (Roche) to generate pEGFP-h Rev7 and pEGFP-m Rev7R. .. PCR products were purified using PCR purification kit (Roche) and then subjected to BP (BP-clonase II, Invitrogen) and LR (LR-clonase II, Invitrogen) reaction according to manufacturer’s instructions.

Article Title: DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes
Article Snippet: DNA was then purified by phenol extraction and ethanol precipitation. .. After enzyme inactivation and phenol extraction, the DNA was religated in a 7-ml volume (1,000 U T4 DNA Ligase, Roche).

Article Title: Episomal HBV persistence within transcribed host nuclear chromatin compartments involves HBx
Article Snippet: .. Digested samples were purified, diluted and then proximity-ligated at 16 °C using T4 DNA ligase (Roche). ..

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism
Article Snippet: The reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. .. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions.

Article Title: Molecular Basis of Aquaporin-7 Permeability Regulation by pH
Article Snippet: .. The purified product was cloned into the corresponding restriction sites of pUG35 digested with the same restriction enzymes, behind the MET25 promoter and in frame with GFP sequence and CYC1-T terminator, using T4 DNA Ligase (Roche, Basel, Switzerland). .. Cloning was performed according to standard protocols [ ] to construct the expression plasmid, pUG35-hAQP7.

Article Title: In silico-guided engineering of Pseudomonas putida towards growth under micro-oxic conditions
Article Snippet: .. DNA fragments were purified from agarose gel using the KG Gel Purification Kit (Machery-Nagel GmbH and Co. Düren, Germany) and ligated into the pSEVA638 backbone (Standardized SEVA plasmid system [ , ]) using T4 DNA Ligase (Roche Applied Science, Indianapolis, IN USA). .. Acetate kinase (ackA ) was isolated from E. coli , the two-subunit gene cluster class I dihydroorotate hydrogenase (pyrK-pyrD B ) and the two-subunit gene cluster class III ribonucleotide triphosphate reductase (nrdD-nrdG ) were ordered using the gene from L. lactis as a template but codon-optimized for P. putida KT2440.

Polymerase Chain Reaction:

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction
Article Snippet: In a 1.5 ml microfuge tube, 5 µg of the purified PCR product was mixed with 10 µl of 10X NEB 2 buffer, 2.5 µl of 20 mM dTTP (500 µM dTTP final concentration) and the volume was adjusted to 98.5 µl with H2 O. .. Then, 10 µl ligation reactions were set-up in multiple tubes, each containing 1 µl of 10X ligation buffer, 100 ng BsaI-digested vector, 25–100 ng T4 DNA polymerase-treated purified inserts (2–8 fold molar excess) and 1.0 unit of T4 DNA ligase (Roche) and incubated for 16 h at 16°C, 60 min at 37°C and heat inactivated at 65°C for 10 min. For electroporation, the contents of ligation reaction were pooled and 4 µl ligation mixture was directly electroporated into 100 µl of competent E. coli cells (efficiency 7–8×109 /µg) and several such electroporations were performed to obtain a library of ∼108 clones.

Article Title: REV7 counteracts DNA double-strand break resection and impacts PARP inhibition
Article Snippet: .. The PCR product and pEGFP-C1 vector were digested using EcoRI and KpnI and ligated using the T4 DNA Ligase (Roche) to generate pEGFP-h Rev7 and pEGFP-m Rev7R. .. Using pEGFP-m Rev7R as a template, pEGFP-C1 based Rev7 truncated constructs was amplified by PCR using the following primers: Forward primer: 5′-ATAT-GAATTC-G-ATGACCACCCTCACGCGC-3′ Reverse primers: mRev7R (1-55aa): 5′-ATAT-GGTACC-GGACATCTGAACCGGCAC -3′ mRev7R (1-81aa): 5′-ATAT-GGTACC-CTCCACATCGTTCTTCTCCAGG -3′ mRev7R (1-110aa): 5′-ATAT-GGTACC-GATGGACAGCAAGGGAGGC-3′ mRev7R (1-140aa): 5′-ATAT-GGTACC-GTTGTGATCCAGGACAGC -3′ mrev7R (1-183aa): 5′-ATAT-GGTACC-GTCGTGCATGTGGACATCCTG-3′ pEGFP-Rev7 mutants (shRNA resistant) were ordered as gBlocks (IDT) and cloned into the pEGFP-C1 vector using EcoRI and KpnI restriction enzymes and quick ligase (NEB).

Article Title: DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes
Article Snippet: After enzyme inactivation and phenol extraction, the DNA was religated in a 7-ml volume (1,000 U T4 DNA Ligase, Roche). .. Three micrograms of 4C library DNA was amplified with Expand Long template PCR System (Roche).

Article Title: Epigenetic and transcriptional dysregulation of VWA2 associated with a MYC-driven oncogenic program in colorectal cancer
Article Snippet: The digested DNA was ligated to 1.25 ml each of 5 pmol/ml Not I and 50 pmol/ml Mse I adaptor using 1 unit of T4 DNA ligase (Roche) overnight at 16 °C. .. The adaptor-ligated template DNA was PCR amplified using 32 P-labeled Not I and Mse I-C primers.

Article Title: Proline oxidase controls proline, glutamate, and glutamine cellular concentrations in a U87 glioblastoma cell line
Article Snippet: PRODH sequence, two clones with partially overlapping regions (pBlue–IRAKp961I0147Q and pCR-BluntII-TOPO-IRCMp5012A1133D) were identified, containing the 5’- and the 3’-terminus portions of the expected full-length PRODH sequence (the one encoding for human PO, 600 amino acids). .. The fragments of interest were subsequently ligated (T4 DNA ligase, Roche) into pGEMT-easy and the sequence of the final construct was confirmed by automated sequencing.

Article Title: Methylation Landscape of Human Breast Cancer Cells in Response to Dietary Compound Resveratrol
Article Snippet: Heat inactivation was carried out at 65°C for 20 min. MseI fragments were then subjected to linker-mediated PCR as essentially described (Klein, et al., 1999). .. At 15°C, 10 ml MseI fragments, 2 ml of ATP (10 mM) and 2 ml T4-DNA ligase (10 U; Roche, Grenzach-Wyhlen, Germany) were added, and primers and DNA fragments were ligated overnight.

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction
Article Snippet: Single tube method Ten microlitres of PCR product was mixed with 1 µl Exonuclease I-Shrimp Alkaline Phosphatase (Exo-SAP; Affymetrix, Santa Clara, CA, USA) in a 0.2 ml PCR tube, vortexed, centrifuged and incubated in a thermocycler programmed for 60 min incubation at 37°C followed by heat inactivation at 80°C for 20 min, then the tube was held at 4°C. .. The contents were mixed well, centrifuged at 4°C and then placed in a thermocycler for incubation at 15°C for 60 min followed by heat inactivation at 75°C for 20 min. Ligation was performed in a total volume of 10 µl containing 1 µl of 10X ligation buffer (New England Biolabs), 25 ng BsaI-HF-digested vector, 1 µl of T4 DNA Polymerase-treated insert (2–5 fold molar excess relative to the vector) and 1.0 unit of T4 DNA ligase (Roche) for 16 h at 16°C followed by 1 h at 37°C and heat inactivation at 65°C for 10 min. For electroporation, the ligation mixture was diluted ten-fold in water and then 1 µl of the diluted ligation sample (containing 250 pg vector equivalent) was electroporated into 25 µl of E. coli competent cells [BL21 (DE3) RIL, Agilent Technologies, CA, USA; efficiency 3–5×108 /µg].

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism
Article Snippet: Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. .. PCR amplification by using the external primers (italics in ) was performed according to the protocol described in section 1.

Article Title: Molecular Basis of Aquaporin-7 Permeability Regulation by pH
Article Snippet: The amplified product was digested with Spe I and Cla I (Roche Diagnostics® ) and purified using Wizard® SV Gel and PCR Clean-Up System kit (Promega). .. The purified product was cloned into the corresponding restriction sites of pUG35 digested with the same restriction enzymes, behind the MET25 promoter and in frame with GFP sequence and CYC1-T terminator, using T4 DNA Ligase (Roche, Basel, Switzerland).

shRNA:

Article Title: REV7 counteracts DNA double-strand break resection and impacts PARP inhibition
Article Snippet: The PCR product and pEGFP-C1 vector were digested using EcoRI and KpnI and ligated using the T4 DNA Ligase (Roche) to generate pEGFP-h Rev7 and pEGFP-m Rev7R. .. Using pEGFP-m Rev7R as a template, pEGFP-C1 based Rev7 truncated constructs was amplified by PCR using the following primers: Forward primer: 5′-ATAT-GAATTC-G-ATGACCACCCTCACGCGC-3′ Reverse primers: mRev7R (1-55aa): 5′-ATAT-GGTACC-GGACATCTGAACCGGCAC -3′ mRev7R (1-81aa): 5′-ATAT-GGTACC-CTCCACATCGTTCTTCTCCAGG -3′ mRev7R (1-110aa): 5′-ATAT-GGTACC-GATGGACAGCAAGGGAGGC-3′ mRev7R (1-140aa): 5′-ATAT-GGTACC-GTTGTGATCCAGGACAGC -3′ mrev7R (1-183aa): 5′-ATAT-GGTACC-GTCGTGCATGTGGACATCCTG-3′ pEGFP-Rev7 mutants (shRNA resistant) were ordered as gBlocks (IDT) and cloned into the pEGFP-C1 vector using EcoRI and KpnI restriction enzymes and quick ligase (NEB).

Chromatin Immunoprecipitation:

Article Title: DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes
Article Snippet: Paragraph title: Chromosome conformation capture on chip (4C) followed by deep-sequencing ... After enzyme inactivation and phenol extraction, the DNA was religated in a 7-ml volume (1,000 U T4 DNA Ligase, Roche).

Plasmid Preparation:

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction
Article Snippet: .. Then, 10 µl ligation reactions were set-up in multiple tubes, each containing 1 µl of 10X ligation buffer, 100 ng BsaI-digested vector, 25–100 ng T4 DNA polymerase-treated purified inserts (2–8 fold molar excess) and 1.0 unit of T4 DNA ligase (Roche) and incubated for 16 h at 16°C, 60 min at 37°C and heat inactivated at 65°C for 10 min. For electroporation, the contents of ligation reaction were pooled and 4 µl ligation mixture was directly electroporated into 100 µl of competent E. coli cells (efficiency 7–8×109 /µg) and several such electroporations were performed to obtain a library of ∼108 clones. .. Concept of the cloning strategy The cloning strategy described here is based on a combination of techniques involving the use of the type IIs restriction endonuclease BsaI to create a linearized vector with 4 base-long 5′-overhangs, and T4 DNA polymerase treatment of the insert in the presence of a single dNTP to create vector-compatible 4 base-long overhangs.

Article Title: REV7 counteracts DNA double-strand break resection and impacts PARP inhibition
Article Snippet: .. The PCR product and pEGFP-C1 vector were digested using EcoRI and KpnI and ligated using the T4 DNA Ligase (Roche) to generate pEGFP-h Rev7 and pEGFP-m Rev7R. .. Using pEGFP-m Rev7R as a template, pEGFP-C1 based Rev7 truncated constructs was amplified by PCR using the following primers: Forward primer: 5′-ATAT-GAATTC-G-ATGACCACCCTCACGCGC-3′ Reverse primers: mRev7R (1-55aa): 5′-ATAT-GGTACC-GGACATCTGAACCGGCAC -3′ mRev7R (1-81aa): 5′-ATAT-GGTACC-CTCCACATCGTTCTTCTCCAGG -3′ mRev7R (1-110aa): 5′-ATAT-GGTACC-GATGGACAGCAAGGGAGGC-3′ mRev7R (1-140aa): 5′-ATAT-GGTACC-GTTGTGATCCAGGACAGC -3′ mrev7R (1-183aa): 5′-ATAT-GGTACC-GTCGTGCATGTGGACATCCTG-3′ pEGFP-Rev7 mutants (shRNA resistant) were ordered as gBlocks (IDT) and cloned into the pEGFP-C1 vector using EcoRI and KpnI restriction enzymes and quick ligase (NEB).

Article Title: Proline oxidase controls proline, glutamate, and glutamine cellular concentrations in a U87 glioblastoma cell line
Article Snippet: Paragraph title: Expression vector ... The fragments of interest were subsequently ligated (T4 DNA ligase, Roche) into pGEMT-easy and the sequence of the final construct was confirmed by automated sequencing.

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction
Article Snippet: .. The contents were mixed well, centrifuged at 4°C and then placed in a thermocycler for incubation at 15°C for 60 min followed by heat inactivation at 75°C for 20 min. Ligation was performed in a total volume of 10 µl containing 1 µl of 10X ligation buffer (New England Biolabs), 25 ng BsaI-HF-digested vector, 1 µl of T4 DNA Polymerase-treated insert (2–5 fold molar excess relative to the vector) and 1.0 unit of T4 DNA ligase (Roche) for 16 h at 16°C followed by 1 h at 37°C and heat inactivation at 65°C for 10 min. For electroporation, the ligation mixture was diluted ten-fold in water and then 1 µl of the diluted ligation sample (containing 250 pg vector equivalent) was electroporated into 25 µl of E. coli competent cells [BL21 (DE3) RIL, Agilent Technologies, CA, USA; efficiency 3–5×108 /µg]. .. The resulting recombinants were analyzed by colony PCR followed by sequencing using an ABI 3730 DNA sequencer (Life Technologies Corporation, USA).

Article Title: Hypoxia-targeted 131I therapy of hepatocellular cancer after systemic mesenchymal stem cell-mediated sodium iodide symporter gene delivery
Article Snippet: .. NIS cDNA was ligated into the pGL3 expression vector using the T4 DNA Ligase (Roche, Basel, Switzerland). .. For cloning of the pcDNA-ITR-HIF-Cherry plasmid, the HIF-responsive promoter was amplified from the pGL3-HIF-LUC vector and the fluorescent protein mCherry was amplified from the pCAG-Kosak-Cherry vector (a gift from M Rosemann, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany).

Article Title: Molecular Basis of Aquaporin-7 Permeability Regulation by pH
Article Snippet: The purified product was cloned into the corresponding restriction sites of pUG35 digested with the same restriction enzymes, behind the MET25 promoter and in frame with GFP sequence and CYC1-T terminator, using T4 DNA Ligase (Roche, Basel, Switzerland). .. Cloning was performed according to standard protocols [ ] to construct the expression plasmid, pUG35-hAQP7.

Article Title: In silico-guided engineering of Pseudomonas putida towards growth under micro-oxic conditions
Article Snippet: .. DNA fragments were purified from agarose gel using the KG Gel Purification Kit (Machery-Nagel GmbH and Co. Düren, Germany) and ligated into the pSEVA638 backbone (Standardized SEVA plasmid system [ , ]) using T4 DNA Ligase (Roche Applied Science, Indianapolis, IN USA). .. Acetate kinase (ackA ) was isolated from E. coli , the two-subunit gene cluster class I dihydroorotate hydrogenase (pyrK-pyrD B ) and the two-subunit gene cluster class III ribonucleotide triphosphate reductase (nrdD-nrdG ) were ordered using the gene from L. lactis as a template but codon-optimized for P. putida KT2440.

SYBR Green Assay:

Article Title: Episomal HBV persistence within transcribed host nuclear chromatin compartments involves HBx
Article Snippet: Digestion efficiencies were quantified by qPCR using a Corbett Rotor-Gene 6000 qPCR device and Quantitect SYBR Green Master Mix (Qiagen, Hilden, Germany) with primers covering the SpeI restriction site within the cccDNA normalized to a non-digested region. .. Digested samples were purified, diluted and then proximity-ligated at 16 °C using T4 DNA ligase (Roche).

Selection:

Article Title: Hypoxia-targeted 131I therapy of hepatocellular cancer after systemic mesenchymal stem cell-mediated sodium iodide symporter gene delivery
Article Snippet: The expression vector pGL3-HIF-LUC (a gift from F Grässer, Universitätsklinikum des Saarlandes, Homburg, Germany) was modified to contain a synthetic promoter composed of a minimal thymidine kinase promoter with six HIF-responsive elements driving transgene expression and a CMV controlled Bsr2 blasticidin resistance gene for selection of transfected cells. .. NIS cDNA was ligated into the pGL3 expression vector using the T4 DNA Ligase (Roche, Basel, Switzerland).

Agarose Gel Electrophoresis:

Article Title: In silico-guided engineering of Pseudomonas putida towards growth under micro-oxic conditions
Article Snippet: .. DNA fragments were purified from agarose gel using the KG Gel Purification Kit (Machery-Nagel GmbH and Co. Düren, Germany) and ligated into the pSEVA638 backbone (Standardized SEVA plasmid system [ , ]) using T4 DNA Ligase (Roche Applied Science, Indianapolis, IN USA). .. Acetate kinase (ackA ) was isolated from E. coli , the two-subunit gene cluster class I dihydroorotate hydrogenase (pyrK-pyrD B ) and the two-subunit gene cluster class III ribonucleotide triphosphate reductase (nrdD-nrdG ) were ordered using the gene from L. lactis as a template but codon-optimized for P. putida KT2440.

Ethanol Precipitation:

Article Title: DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes
Article Snippet: DNA was then purified by phenol extraction and ethanol precipitation. .. After enzyme inactivation and phenol extraction, the DNA was religated in a 7-ml volume (1,000 U T4 DNA Ligase, Roche).

Next-Generation Sequencing:

Article Title: Not All H3K4 methylations are Created Equal: Mll2/COMPASS Dependency in Primordial Germ Cell Specification
Article Snippet: Samples were then diluted and ligated with T4 DNA ligase (Roche) to generate the 4C template. .. 4C–PCR was performed with the primers in the (NNNNNN indicates location of index sequence) and next generation sequencing was performed on Nextseq 500 (Illumina).

DNA Methylation Assay:

Article Title: Methylation Landscape of Human Breast Cancer Cells in Response to Dietary Compound Resveratrol
Article Snippet: Paragraph title: Genome-wide analysis of DNA methylation by array-PRIMES (aPRIMES) ... At 15°C, 10 ml MseI fragments, 2 ml of ATP (10 mM) and 2 ml T4-DNA ligase (10 U; Roche, Grenzach-Wyhlen, Germany) were added, and primers and DNA fragments were ligated overnight.

Concentration Assay:

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction
Article Snippet: In a 1.5 ml microfuge tube, 5 µg of the purified PCR product was mixed with 10 µl of 10X NEB 2 buffer, 2.5 µl of 20 mM dTTP (500 µM dTTP final concentration) and the volume was adjusted to 98.5 µl with H2 O. .. Then, 10 µl ligation reactions were set-up in multiple tubes, each containing 1 µl of 10X ligation buffer, 100 ng BsaI-digested vector, 25–100 ng T4 DNA polymerase-treated purified inserts (2–8 fold molar excess) and 1.0 unit of T4 DNA ligase (Roche) and incubated for 16 h at 16°C, 60 min at 37°C and heat inactivated at 65°C for 10 min. For electroporation, the contents of ligation reaction were pooled and 4 µl ligation mixture was directly electroporated into 100 µl of competent E. coli cells (efficiency 7–8×109 /µg) and several such electroporations were performed to obtain a library of ∼108 clones.

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction
Article Snippet: One microlitre of dTTP (20 mM; 1.7 mM final concentration) was added to each tube containing the Exo-SAP treated inserts, mixed well by mild vortexing followed by brief centrifugation at 4°C, and 1.5 units (0.5 µl) of T4 DNA polymerase (New England Biolabs) were added. .. The contents were mixed well, centrifuged at 4°C and then placed in a thermocycler for incubation at 15°C for 60 min followed by heat inactivation at 75°C for 20 min. Ligation was performed in a total volume of 10 µl containing 1 µl of 10X ligation buffer (New England Biolabs), 25 ng BsaI-HF-digested vector, 1 µl of T4 DNA Polymerase-treated insert (2–5 fold molar excess relative to the vector) and 1.0 unit of T4 DNA ligase (Roche) for 16 h at 16°C followed by 1 h at 37°C and heat inactivation at 65°C for 10 min. For electroporation, the ligation mixture was diluted ten-fold in water and then 1 µl of the diluted ligation sample (containing 250 pg vector equivalent) was electroporated into 25 µl of E. coli competent cells [BL21 (DE3) RIL, Agilent Technologies, CA, USA; efficiency 3–5×108 /µg].

Article Title: DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes
Article Snippet: To sequester SDS, Triton X-100 was then added to a final concentration of 1.8%. .. After heat inactivation (65 °C for 30 min), the reaction was diluted to a final volume of 7 ml with ligation buffer containing 100 U T4 DNA Ligase (Roche) and incubated at 16 °C overnight.

Gel Purification:

Article Title: In silico-guided engineering of Pseudomonas putida towards growth under micro-oxic conditions
Article Snippet: .. DNA fragments were purified from agarose gel using the KG Gel Purification Kit (Machery-Nagel GmbH and Co. Düren, Germany) and ligated into the pSEVA638 backbone (Standardized SEVA plasmid system [ , ]) using T4 DNA Ligase (Roche Applied Science, Indianapolis, IN USA). .. Acetate kinase (ackA ) was isolated from E. coli , the two-subunit gene cluster class I dihydroorotate hydrogenase (pyrK-pyrD B ) and the two-subunit gene cluster class III ribonucleotide triphosphate reductase (nrdD-nrdG ) were ordered using the gene from L. lactis as a template but codon-optimized for P. putida KT2440.

Lysis:

Article Title: Episomal HBV persistence within transcribed host nuclear chromatin compartments involves HBx
Article Snippet: Briefly, interacting DNA segments were cross-linked using 1% formaldehyde and nuclei were isolated using a lysis buffer containing 0.4% Tergitol (NP-40 substitute) followed by incubation at 37 °C in the presence of 0.3% SDS. .. Digested samples were purified, diluted and then proximity-ligated at 16 °C using T4 DNA ligase (Roche).

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    Roche t4 dna ligase
    AFM images of DNA-DNA crossings. (a) Bare DNA (3.8 kbp). (b) and (c) DNA with <t>T4</t> DNA ligase andATP. Solid arrows indicate higher crossings consistent with ligase binding, outlined arrows indicateshallower crossings consistent with bare DNA.
    T4 Dna Ligase, supplied by Roche, used in various techniques. Bioz Stars score: 97/100, based on 278 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    AFM images of DNA-DNA crossings. (a) Bare DNA (3.8 kbp). (b) and (c) DNA with T4 DNA ligase andATP. Solid arrows indicate higher crossings consistent with ligase binding, outlined arrows indicateshallower crossings consistent with bare DNA.

    Journal: Biomicrofluidics

    Article Title: Probing transient protein-mediated DNA linkages using nanoconfinement

    doi: 10.1063/1.4882775

    Figure Lengend Snippet: AFM images of DNA-DNA crossings. (a) Bare DNA (3.8 kbp). (b) and (c) DNA with T4 DNA ligase andATP. Solid arrows indicate higher crossings consistent with ligase binding, outlined arrows indicateshallower crossings consistent with bare DNA.

    Article Snippet: For measurements with T4 DNA ligase and ATP, the respective finalconcentrations were 5 URoche /ml (Roche) and 1 mM, respectively.

    Techniques: Binding Assay

    Mean aligned DNA molecule loop lengths as function of time for 22 molecules per dataset withtheir linear fits. Bare λ-DNA (blue), λ-DNA with T4 DNA ligase (green), and λ-DNA with T4 DNA ligaseand ATP (red).

    Journal: Biomicrofluidics

    Article Title: Probing transient protein-mediated DNA linkages using nanoconfinement

    doi: 10.1063/1.4882775

    Figure Lengend Snippet: Mean aligned DNA molecule loop lengths as function of time for 22 molecules per dataset withtheir linear fits. Bare λ-DNA (blue), λ-DNA with T4 DNA ligase (green), and λ-DNA with T4 DNA ligaseand ATP (red).

    Article Snippet: For measurements with T4 DNA ligase and ATP, the respective finalconcentrations were 5 URoche /ml (Roche) and 1 mM, respectively.

    Techniques:

    Histogram of end-to-end lengths of extended DNA molecules, bare λ-DNA (solid bars), λ-DNA with T4DNA ligase (gray bars), and λ-DNA with T4 DNA ligase and ATP (white bars). A Gaussian was fit toeach distribution to determine the

    Journal: Biomicrofluidics

    Article Title: Probing transient protein-mediated DNA linkages using nanoconfinement

    doi: 10.1063/1.4882775

    Figure Lengend Snippet: Histogram of end-to-end lengths of extended DNA molecules, bare λ-DNA (solid bars), λ-DNA with T4DNA ligase (gray bars), and λ-DNA with T4 DNA ligase and ATP (white bars). A Gaussian was fit toeach distribution to determine the

    Article Snippet: For measurements with T4 DNA ligase and ATP, the respective finalconcentrations were 5 URoche /ml (Roche) and 1 mM, respectively.

    Techniques:

    Histograms of heights of DNA-DNA crossings. (a) Bare DNA (N = 41). (b) DNA with T4 DNA ligase andATP (N = 174). The red dotted line corresponds to unoccupied crossings, the blue dashed line tooccupied crossings, and the

    Journal: Biomicrofluidics

    Article Title: Probing transient protein-mediated DNA linkages using nanoconfinement

    doi: 10.1063/1.4882775

    Figure Lengend Snippet: Histograms of heights of DNA-DNA crossings. (a) Bare DNA (N = 41). (b) DNA with T4 DNA ligase andATP (N = 174). The red dotted line corresponds to unoccupied crossings, the blue dashed line tooccupied crossings, and the

    Article Snippet: For measurements with T4 DNA ligase and ATP, the respective finalconcentrations were 5 URoche /ml (Roche) and 1 mM, respectively.

    Techniques:

    ( A ) Schematic of the device. ( B,C ) Kymographs showing the fluorescent intensity along nanochannel axis as a function of time for bare λ -DNA ( B ) and λ -DNA with T4 DNA ligase in a catalytically active buffer ( C ), respectively. ( D ) Center of mass of the molecules in ( B ) (red, diamonds) and ( C ) (blue, circles) as function of time.

    Journal: Scientific Reports

    Article Title: Motor-like DNA motion due to an ATP-hydrolyzing protein under nanoconfinement

    doi: 10.1038/s41598-018-28278-0

    Figure Lengend Snippet: ( A ) Schematic of the device. ( B,C ) Kymographs showing the fluorescent intensity along nanochannel axis as a function of time for bare λ -DNA ( B ) and λ -DNA with T4 DNA ligase in a catalytically active buffer ( C ), respectively. ( D ) Center of mass of the molecules in ( B ) (red, diamonds) and ( C ) (blue, circles) as function of time.

    Article Snippet: This solution was incubated for 1 hour at 16 °C to allow full equilibrization of Mg2+ , ATP, and T4 DNA ligase.

    Techniques:

    Average mean-square displacement curves calculated from the center of mass position for 20 molecules for each condition. Blue circles correspond to data DNA with T4 DNA ligase in the presence of its cofactors, while red diamonds indicate bare DNA. Error bars are the standard deviation between molecules of the experimental set, and are depicted only for select data points to illustrate the trend. The continuous and dashed curves correspond to the fits for the conditions as described in the text.

    Journal: Scientific Reports

    Article Title: Motor-like DNA motion due to an ATP-hydrolyzing protein under nanoconfinement

    doi: 10.1038/s41598-018-28278-0

    Figure Lengend Snippet: Average mean-square displacement curves calculated from the center of mass position for 20 molecules for each condition. Blue circles correspond to data DNA with T4 DNA ligase in the presence of its cofactors, while red diamonds indicate bare DNA. Error bars are the standard deviation between molecules of the experimental set, and are depicted only for select data points to illustrate the trend. The continuous and dashed curves correspond to the fits for the conditions as described in the text.

    Article Snippet: This solution was incubated for 1 hour at 16 °C to allow full equilibrization of Mg2+ , ATP, and T4 DNA ligase.

    Techniques: Standard Deviation