t4 ligase  (Roche)

 
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    T4 ligase
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    Restriction enzyme cloning in th
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    Structured Review

    Roche t4 ligase

    https://www.bioz.com/result/t4 ligase/product/Roche
    Average 97 stars, based on 23 article reviews
    Price from $9.99 to $1999.99
    t4 ligase - by Bioz Stars, 2019-12
    97/100 stars

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    Related Articles

    Clone Assay:

    Article Title: A Fundamental Regulatory Mechanism Operating through OmpR and DNA Topology Controls Expression of Salmonella Pathogenicity Islands SPI-1 and SPI-2
    Article Snippet: Paragraph title: Cloning ... SmaI and XbaI digested amplicons were spin column purified, then ligated to pZep or pZec by T4 ligase (Roche).

    Article Title: Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines
    Article Snippet: The resulting 0.7 kb amplicon was then purified from the agarose gel (Qiagen DNA extraction kit, Hilden, Germany) and subsequently ligated (T4 ligase, Roche, Basel, Switzerland) into the LentiCRISPR Puro V2 at the site of the discarded puromycin fragment (1.3 kb). .. PCR conditions, using a Pfu DNA polymerase (Invitrogen, Carlsbad, CA, USA), were as follows: 95 °C for 5 min, 40 cycles of 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 0.5 min, with a final extension at 72 °C for 10 min.

    Article Title: Production, active staining and gas chromatography assay analysis of recombinant aminopeptidase P from Lactococcus lactis ssp. lactis DSM 20481
    Article Snippet: Paragraph title: Cloning, construction of expression vectors and sequencing of pepP ... Ligations were performed according to manufacturer’s (Promega) protocols using T4-ligase (Roche).

    Article Title: Crystal Structure of the NS3 Protease-Helicase from Dengue Virus
    Article Snippet: Den4 NS3hel (residues 177 to 618) was cloned from the full-length NS3 gene by PCR using forward primer 5′-GAAGTGGATGAGGACATT-3′ and reverse primer 5′-GTGGTG CTCGAG TTACTTTCTTCCACTGGCAAA-3′ (the XhoI site is underlined). .. The PCR fragment was digested with XhoI and cloned into a modified pET32b plasmid using T4 ligase (Roche). .. The expression of the Den4 NS3hel protein was done using the same protocol as that described above for scNS2B18 NS3.

    Article Title: Differential expression of alphaB-crystallin and Hsp27-1 in anaplastic thyroid carcinomas because of tumor-specific alphaB-crystallin gene (CRYAB) silencing
    Article Snippet: BamH1, EcoR1, Boehringer Mannheim chemoluminescence blotting substrate, T4 deoxyribonucleic acid (DNA) ligase, and TRIzol® reagent were obtained from Roche Diagnostics (Mannheim, Germany). .. BamH1, EcoR1, Boehringer Mannheim chemoluminescence blotting substrate, T4 deoxyribonucleic acid (DNA) ligase, and TRIzol® reagent were obtained from Roche Diagnostics (Mannheim, Germany).

    Centrifugation:

    Article Title: N-myc Modulates Expression of p73 in Neuroblastoma
    Article Snippet: Isolated chromosomes were stained with two fluorochromes, Hoechst 33258 and chromomycin A3 (Calbiochem, La Jolla, CA), and allowed to stand for 48 hours using a combination of slow centrifugation and sedimentation. .. Chromosome 1 genomic DNA was digested first with Not I and ligated to the Not I and Eco RV digested plasmid vector Bluescript SK+ (Strategene, La Jolla, CA) with T4 ligase (Roche Molecular Biochemicals, Indianapolis, IN).

    Article Title: Ribonucleoprotein Assembly Defects Correlate with Spinal Muscular Atrophy Severity and Preferentially Affect a Subset of Spliceosomal snRNPs
    Article Snippet: After five washes with the same buffer, bound RNAs were recovered from immunoprecipitates by proteinase K treatment, phenol/chloroform extraction and ethanol precipitation. .. 3′-end labeling experiments were carried out in the presence of [32 P] pCp (3000Ci/mmol) and T4 RNA ligase (Roche) following manufacturer's instructions, and unincorporated nucleotides were removed by centrifugation through micro Bio-Spin P-30 columns (Biorad). .. RNAs were analyzed by electrophoresis on 6% polyacrylamide/8M urea gels and autoradiography.

    Amplification:

    Article Title: DNA-Protein Vaccination Strategy Does Not Protect from Challenge with African Swine Fever Virus Armenia 2007 Strain
    Article Snippet: The fragments were amplified by PCR using the specific primers for each gene from lysate of cells infected with the Ba71V strain (GenBank: NC_001659.2), with the exception of p72 which was amplified from cells infected with ASFV E70 (GenBank: AY578692.1). .. PCR products and pcDNA vector were digested with the corresponding restriction enzymes and ligated with T4 ligase (Roche).

    Article Title: Negative Feedback and Transcriptional Overshooting in a Regulatory Network for Horizontal Gene Transfer
    Article Snippet: PCR amplification was carried out with Vent DNA polymerase (Biolabs) and consisted of 95°C for 10 min, then 28 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s and a final step of 72°C for 5 min. PCR products were digested with XhoI and BamHI at 37°C for 2 h and the products purified using QIAquick Gel Extraction Kit (Qiagen). .. Digested and purified fragments were ligated into pUA66 plasmid DNA using T4 ligase (Roche) with overnight incubatio n at 16°C.

    Article Title: A Fundamental Regulatory Mechanism Operating through OmpR and DNA Topology Controls Expression of Salmonella Pathogenicity Islands SPI-1 and SPI-2
    Article Snippet: Gene promoter sequences were PCR amplified using the Phusion DNA polymerase (NEB) and primers listed in . .. SmaI and XbaI digested amplicons were spin column purified, then ligated to pZep or pZec by T4 ligase (Roche).

    Article Title: Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines
    Article Snippet: To generate LCmCherry V2, the mCherry sequence was obtained from FU-mCherry-w (derived from FUGW) [ ] and then digested with Bsi WI and Sac II restriction enzymes (New England Biolabs, Ipswich, MA, USA). .. The resulting 0.7 kb amplicon was then purified from the agarose gel (Qiagen DNA extraction kit, Hilden, Germany) and subsequently ligated (T4 ligase, Roche, Basel, Switzerland) into the LentiCRISPR Puro V2 at the site of the discarded puromycin fragment (1.3 kb). .. Secondly, the full length U6 promoter from zebrafish (U6ZF) was amplified by PCR from genomic DNA Danio rerio , using FwU6ZF and RvU6Zf primers.

    Article Title: Production, active staining and gas chromatography assay analysis of recombinant aminopeptidase P from Lactococcus lactis ssp. lactis DSM 20481
    Article Snippet: The amplified pepP gene PCR products were purified (QIAquick Gel Extraction Kit) after electrophoresis in an agarose gel [1 % (w/v)]. .. Ligations were performed according to manufacturer’s (Promega) protocols using T4-ligase (Roche).

    Article Title: Distinct Amino Termini of Two Human HCS Isoforms Influence Biotin Acceptor Substrate Recognition
    Article Snippet: The coding sequence for 58-HCS was amplified from a pGEX construct provided by Dr. Roy Gravel. .. The FL-HCS cDNA sequence (GenBankTM accession number ) was purchased from OriGene Technologies, Inc. After digestion of the PCR product with Esp3I (Fermentas), the resulting two coding sequence fragments were ligated into the linearized pSUMO vector using T4 ligase (Roche Applied Science), and the ligation mixtures were transformed into the E. coli top10 strain.

    Article Title: The ClgR Protein Regulates Transcription of the clpP Operon in Bifidobacterium breve UCC 2003
    Article Snippet: The clgR gene from B. breve UCC 2003 was amplified using the primers 903-uni and 903-rev, which contain a BamHI and a HindIII restriction site, respectively. .. The resultant 566-bp PCR fragment was digested with BamHI and HindIII and ligated into similarly restricted pQE30, using the T4 DNA ligase enzyme (Roche, Sussex, United Kingdom), to generate plasmid pQE-ClgR, which was introduced into E. coli M15 (QIAGEN, United Kingdom) as described by Sambrook et al. ( ).

    Article Title: Genetic and Transcriptional Organization of the clpC Locus in Bifidobacterium breve UCC 2003
    Article Snippet: Each PCR cycling profile consisted of an initial denaturation step of 5 min at 95°C, followed by 35 cycles of amplification as follows: denaturation for 30 s at 95°C, annealing for 30 s at 55°C, and extension for 1 min at 72°C. .. The resultant 566-bp PCR fragment was digested with BamHI (Roche, Sussex, United Kingdom) and HindIII (Roche, Sussex, United Kingdom) and ligated into similarly restricted pQE30 using the T4 DNA ligase enzyme (Roche) to generate plasmid pQE-ClgR, which was introduced by electrotransformation into E. coli M15 (QIAGEN, United Kingdom) as described by Sambrook and Russell ( ).

    Construct:

    Article Title: DNA-Protein Vaccination Strategy Does Not Protect from Challenge with African Swine Fever Virus Armenia 2007 Strain
    Article Snippet: PCR products and pcDNA vector were digested with the corresponding restriction enzymes and ligated with T4 ligase (Roche). .. PCR products and pcDNA vector were digested with the corresponding restriction enzymes and ligated with T4 ligase (Roche).

    Article Title: A Fundamental Regulatory Mechanism Operating through OmpR and DNA Topology Controls Expression of Salmonella Pathogenicity Islands SPI-1 and SPI-2
    Article Snippet: Transcriptional reporter fusions were constructed by cloning gene promoters in pZep and pZec vectors, which contain a promoterless gfp + gene , . .. SmaI and XbaI digested amplicons were spin column purified, then ligated to pZep or pZec by T4 ligase (Roche).

    Article Title: Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions
    Article Snippet: All integrin-GFP constructs were made using three-segment (A, B, C) overlap PCR with wild-type human αV cDNA and either pEGFP-N1 (Clontech, for ɑV-GFP–less-constrained) or moxGFP (for ɑV-GFP–constrained) as sources for GFP cDNA. .. After the three segments had been made and joined together using PCR (Accuprime Pfx, high-fidelity polymerase; Thermo Fisher), the complete A–C sequence and the wild-type ɑV-pcDNA3.1 plasmid were cut with restriction enzymes (New England Biolabs) and ligated together with T4 ligase (Roche) after dephosphorylation (rAPID alkaline phosphatase; Roche) and purification (Qiagen) of the linearized plasmid.

    Article Title: Distinct Amino Termini of Two Human HCS Isoforms Influence Biotin Acceptor Substrate Recognition
    Article Snippet: The coding sequence for 58-HCS was amplified from a pGEX construct provided by Dr. Roy Gravel. .. The FL-HCS cDNA sequence (GenBankTM accession number ) was purchased from OriGene Technologies, Inc. After digestion of the PCR product with Esp3I (Fermentas), the resulting two coding sequence fragments were ligated into the linearized pSUMO vector using T4 ligase (Roche Applied Science), and the ligation mixtures were transformed into the E. coli top10 strain.

    Article Title: Crystal Structure of the NS3 Protease-Helicase from Dengue Virus
    Article Snippet: The expressed construct thus comprises the thioredoxin (Trx) protein followed by a hexahistidine tag and a thrombin cleavage site fused at the N terminus of scNS2B18 NS3. .. The PCR fragment was digested with XhoI and cloned into a modified pET32b plasmid using T4 ligase (Roche).

    Article Title: Isolation and Transcription Profiling of Low-O2 Stress-Associated cDNA Clones from the Flooding-stress-tolerant FR13A Rice Genotype
    Article Snippet: SSL was constructed using the mRNA pool from FR13A low O2 -stressed roots as the tester and the mRNA population from FR13A control roots as the driver. .. First strand cDNA was purified using Qiaquick columns (Qiagen) and specific oligonucleotides (with sequences 5′-GCTAGCATATGGGCCCGAATTCC-3′ for the tester and 5′-CCCTTTAGTGAGGGTTAATTTC-3′ for the driver) were ligated at the 3′ end of the respective cDNA populations using T4 RNA ligase (Roche).

    Electrophoresis:

    Article Title: Production, active staining and gas chromatography assay analysis of recombinant aminopeptidase P from Lactococcus lactis ssp. lactis DSM 20481
    Article Snippet: The amplified pepP gene PCR products were purified (QIAquick Gel Extraction Kit) after electrophoresis in an agarose gel [1 % (w/v)]. .. Ligations were performed according to manufacturer’s (Promega) protocols using T4-ligase (Roche).

    Article Title: Identification of a Novel Streptococcal Gene Cassette Mediating SOS Mutagenesis in Streptococcus uberis
    Article Snippet: Following electrophoresis, gels were scanned with a Fuji FLA-5100 scanner (Fuji Photo Film Co., Ltd., Japan) using an excitation laser at 532 nm, an output voltage of 400 V, and an LPG emission filter. .. Oligonucleotide pairs p26/p27 and p28/p29 with overhangs creating EcoRI and XbaI compatible ends, respectively, were annealed, treated with T4 polynucleotide kinase (MBI Fermentas), and ligated with T4 ligase (Roche) to XbaI-EcoRI cut pBluescript-II SK+ to obtain pBluescript-IR and pBluescript-ctrl, respectively.

    Incubation:

    Article Title: Identification of a Novel Streptococcal Gene Cassette Mediating SOS Mutagenesis in Streptococcus uberis
    Article Snippet: Gel shift reactions were incubated at 25°C for 15 min followed by electrophoresis on a 5% polyacrylamide gel with 0.5× TBE (0.0445 M Tris, 0.0455 M borate, 1.25 M EDTA) at room temperature. .. Oligonucleotide pairs p26/p27 and p28/p29 with overhangs creating EcoRI and XbaI compatible ends, respectively, were annealed, treated with T4 polynucleotide kinase (MBI Fermentas), and ligated with T4 ligase (Roche) to XbaI-EcoRI cut pBluescript-II SK+ to obtain pBluescript-IR and pBluescript-ctrl, respectively.

    Expressing:

    Article Title: DNA-Protein Vaccination Strategy Does Not Protect from Challenge with African Swine Fever Virus Armenia 2007 Strain
    Article Snippet: The pcDNA 3.1 plasmids expressing ASFV-specific genes were prepared as described [ ]. .. PCR products and pcDNA vector were digested with the corresponding restriction enzymes and ligated with T4 ligase (Roche).

    Article Title: A Fundamental Regulatory Mechanism Operating through OmpR and DNA Topology Controls Expression of Salmonella Pathogenicity Islands SPI-1 and SPI-2
    Article Snippet: The removal of the cat gene from pZep to generate pZec had no effect on expression of cloned promoters (not shown). .. SmaI and XbaI digested amplicons were spin column purified, then ligated to pZep or pZec by T4 ligase (Roche).

    Article Title: Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines
    Article Snippet: The expression vector LentiCRISPR-Cas9-2A-mCherryU6ZF (LcU6ZF, hereafter) created for fish cell lines was based on the mammalian LentiCRISPR Puro V2 from Feng Zhang´s lab, (addgene plasmid #52961) [ ] which was modified in two steps, as follows. .. The resulting 0.7 kb amplicon was then purified from the agarose gel (Qiagen DNA extraction kit, Hilden, Germany) and subsequently ligated (T4 ligase, Roche, Basel, Switzerland) into the LentiCRISPR Puro V2 at the site of the discarded puromycin fragment (1.3 kb).

    Article Title: Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions
    Article Snippet: Paragraph title: cDNA Expression Vectors. ... After the three segments had been made and joined together using PCR (Accuprime Pfx, high-fidelity polymerase; Thermo Fisher), the complete A–C sequence and the wild-type ɑV-pcDNA3.1 plasmid were cut with restriction enzymes (New England Biolabs) and ligated together with T4 ligase (Roche) after dephosphorylation (rAPID alkaline phosphatase; Roche) and purification (Qiagen) of the linearized plasmid.

    Article Title: Production, active staining and gas chromatography assay analysis of recombinant aminopeptidase P from Lactococcus lactis ssp. lactis DSM 20481
    Article Snippet: Paragraph title: Cloning, construction of expression vectors and sequencing of pepP ... Ligations were performed according to manufacturer’s (Promega) protocols using T4-ligase (Roche).

    Article Title: Distinct Amino Termini of Two Human HCS Isoforms Influence Biotin Acceptor Substrate Recognition
    Article Snippet: Paragraph title: Expression Plasmid Construction ... The FL-HCS cDNA sequence (GenBankTM accession number ) was purchased from OriGene Technologies, Inc. After digestion of the PCR product with Esp3I (Fermentas), the resulting two coding sequence fragments were ligated into the linearized pSUMO vector using T4 ligase (Roche Applied Science), and the ligation mixtures were transformed into the E. coli top10 strain.

    Article Title: Crystal Structure of the NS3 Protease-Helicase from Dengue Virus
    Article Snippet: Paragraph title: Cloning and expression. ... The PCR fragment was digested with XhoI and cloned into a modified pET32b plasmid using T4 ligase (Roche).

    Modification:

    Article Title: Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines
    Article Snippet: The expression vector LentiCRISPR-Cas9-2A-mCherryU6ZF (LcU6ZF, hereafter) created for fish cell lines was based on the mammalian LentiCRISPR Puro V2 from Feng Zhang´s lab, (addgene plasmid #52961) [ ] which was modified in two steps, as follows. .. The resulting 0.7 kb amplicon was then purified from the agarose gel (Qiagen DNA extraction kit, Hilden, Germany) and subsequently ligated (T4 ligase, Roche, Basel, Switzerland) into the LentiCRISPR Puro V2 at the site of the discarded puromycin fragment (1.3 kb).

    Article Title: Crystal Structure of the NS3 Protease-Helicase from Dengue Virus
    Article Snippet: Den4 NS3hel (residues 177 to 618) was cloned from the full-length NS3 gene by PCR using forward primer 5′-GAAGTGGATGAGGACATT-3′ and reverse primer 5′-GTGGTG CTCGAG TTACTTTCTTCCACTGGCAAA-3′ (the XhoI site is underlined). .. The PCR fragment was digested with XhoI and cloned into a modified pET32b plasmid using T4 ligase (Roche). .. The expression of the Den4 NS3hel protein was done using the same protocol as that described above for scNS2B18 NS3.

    Transformation Assay:

    Article Title: Distinct Amino Termini of Two Human HCS Isoforms Influence Biotin Acceptor Substrate Recognition
    Article Snippet: The coding sequence for 58-HCS was amplified from a pGEX construct provided by Dr. Roy Gravel. .. The FL-HCS cDNA sequence (GenBankTM accession number ) was purchased from OriGene Technologies, Inc. After digestion of the PCR product with Esp3I (Fermentas), the resulting two coding sequence fragments were ligated into the linearized pSUMO vector using T4 ligase (Roche Applied Science), and the ligation mixtures were transformed into the E. coli top10 strain. .. The plasmids encoding His6 -SUMO-FL-HCS and His6 -SUMO-58-HCS were transformed into E. coli RosettaTM (DE3).

    Article Title: Roles of the Essential Protein FtsA in Cell Growth and Division in Streptococcus pneumoniae
    Article Snippet: Standard protocols for molecular cloning, transformation, and DNA analysis were used ( ). .. Restriction enzymes, DNA polymerase, and T4 ligase were purchased from Roche Diagnostics or New England BioLabs.

    Article Title: Crystal Structure of the NS3 Protease-Helicase from Dengue Virus
    Article Snippet: Transformed Escherichia coli BL21-CodonPlus clones (Stratagene) were grown at 18°C in an autoinduction medium ( ) supplemented with 100 μg ml−1 ampicillin and 50 μg ml−1 chloramphenicol. .. The PCR fragment was digested with XhoI and cloned into a modified pET32b plasmid using T4 ligase (Roche).

    Derivative Assay:

    Article Title: Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines
    Article Snippet: To generate LCmCherry V2, the mCherry sequence was obtained from FU-mCherry-w (derived from FUGW) [ ] and then digested with Bsi WI and Sac II restriction enzymes (New England Biolabs, Ipswich, MA, USA). .. The resulting 0.7 kb amplicon was then purified from the agarose gel (Qiagen DNA extraction kit, Hilden, Germany) and subsequently ligated (T4 ligase, Roche, Basel, Switzerland) into the LentiCRISPR Puro V2 at the site of the discarded puromycin fragment (1.3 kb).

    Countercurrent Chromatography:

    Article Title: Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions
    Article Snippet: After the three segments had been made and joined together using PCR (Accuprime Pfx, high-fidelity polymerase; Thermo Fisher), the complete A–C sequence and the wild-type ɑV-pcDNA3.1 plasmid were cut with restriction enzymes (New England Biolabs) and ligated together with T4 ligase (Roche) after dephosphorylation (rAPID alkaline phosphatase; Roche) and purification (Qiagen) of the linearized plasmid. .. After the three segments had been made and joined together using PCR (Accuprime Pfx, high-fidelity polymerase; Thermo Fisher), the complete A–C sequence and the wild-type ɑV-pcDNA3.1 plasmid were cut with restriction enzymes (New England Biolabs) and ligated together with T4 ligase (Roche) after dephosphorylation (rAPID alkaline phosphatase; Roche) and purification (Qiagen) of the linearized plasmid.

    Electroporation:

    Article Title: N-myc Modulates Expression of p73 in Neuroblastoma
    Article Snippet: Chromosome 1 genomic DNA was digested first with Not I and ligated to the Not I and Eco RV digested plasmid vector Bluescript SK+ (Strategene, La Jolla, CA) with T4 ligase (Roche Molecular Biochemicals, Indianapolis, IN). .. Chromosome 1 genomic DNA was digested first with Not I and ligated to the Not I and Eco RV digested plasmid vector Bluescript SK+ (Strategene, La Jolla, CA) with T4 ligase (Roche Molecular Biochemicals, Indianapolis, IN).

    Electro Cell Manipulation:

    Article Title: Insights into Lantibiotic Immunity Provided by Bioengineering of LtnI
    Article Snippet: In both cases, electrotransformation was performed with an Electro cell manipulator (BTX-Harvard apparatus). .. DNA ligations were executed according to established procedures using T4 ligase supplied by Roche Diagnostics.

    Inverse PCR:

    Article Title: Influence of IS256 on Genome Variability and Formation of Small-Colony Variants in Staphylococcus aureus
    Article Snippet: The localizations of IS 256 and recombinant IS 256 elements in different insertion mutants of S. aureus HG001 were determined by inverse PCR as previously described ( ) ( ). .. After circularization of the fragments using the T4 ligase (Roche Applied Science GmbH, Mannheim, Germany), the insertion sites were detected by PCR analysis using the outgoing primers IS91rev and IS141for, which anneal within the 5′ end of IS 256 ( ).

    Chromatography:

    Article Title: Human coagulation factor VIII domain-specific recombinant polypeptide expression
    Article Snippet: Bam HI and Xho I were purchased from Fermentas (Vilnius, Lithuania), and T4 ligase was obtained from Roche (Indianapolis, IN, USA). .. Bam HI and Xho I were purchased from Fermentas (Vilnius, Lithuania), and T4 ligase was obtained from Roche (Indianapolis, IN, USA).

    Ligation:

    Article Title: Novel Methods for Genetic Transformation of Natural Bacillus subtilis Isolates Used To Study the Regulation of the Mycosubtilin and Surfactin Synthetases
    Article Snippet: After 20 min, 0.3 ml of TY medium was added, and growth was continued for another 30 min, after which the cells were plated on selective TY-agar plates. .. All ligation reactions were performed overnight at room temperature, using T4 ligase and buffer from Roche Diagnostics in a total volume of 30 μl. .. For polyethylene glycol (PEG) ligations, a PEG 8000 solution (heat sterilized) was added to a final concentration of 15% ( ).

    Article Title: Distinct Amino Termini of Two Human HCS Isoforms Influence Biotin Acceptor Substrate Recognition
    Article Snippet: The coding sequence for 58-HCS was amplified from a pGEX construct provided by Dr. Roy Gravel. .. The FL-HCS cDNA sequence (GenBankTM accession number ) was purchased from OriGene Technologies, Inc. After digestion of the PCR product with Esp3I (Fermentas), the resulting two coding sequence fragments were ligated into the linearized pSUMO vector using T4 ligase (Roche Applied Science), and the ligation mixtures were transformed into the E. coli top10 strain. .. The plasmids encoding His6 -SUMO-FL-HCS and His6 -SUMO-58-HCS were transformed into E. coli RosettaTM (DE3).

    Protease Inhibitor:

    Article Title: Ribonucleoprotein Assembly Defects Correlate with Spinal Muscular Atrophy Severity and Preferentially Affect a Subset of Spliceosomal snRNPs
    Article Snippet: Immunoprecipitations with anti-Sm (Y12) antibodies from snRNP assembly reactions or whole tissue extracts (200 µg) were carried out in RSB-500 buffer (500 mM NaCl, 10 mM Tris-HCl pH 7.4, 2.5 mM MgCl2 ) containing 0.1% NP40, EDTA-free protease inhibitor cocktail (Roche) and phosphatase inhibitors (50 mM NaF, 0.2 mM Na3 VO4 ) for 2 h at 4°C. .. 3′-end labeling experiments were carried out in the presence of [32 P] pCp (3000Ci/mmol) and T4 RNA ligase (Roche) following manufacturer's instructions, and unincorporated nucleotides were removed by centrifugation through micro Bio-Spin P-30 columns (Biorad).

    RLGS:

    Article Title: N-myc Modulates Expression of p73 in Neuroblastoma
    Article Snippet: Chromosome 1 genomic DNA was digested first with Not I and ligated to the Not I and Eco RV digested plasmid vector Bluescript SK+ (Strategene, La Jolla, CA) with T4 ligase (Roche Molecular Biochemicals, Indianapolis, IN). .. The circular DNA was introduced into Epicurian coli, XL1-blue MRF′ cells by electroporation according to the conditions recommended by the manufacturer.

    Infection:

    Article Title: DNA-Protein Vaccination Strategy Does Not Protect from Challenge with African Swine Fever Virus Armenia 2007 Strain
    Article Snippet: The fragments were amplified by PCR using the specific primers for each gene from lysate of cells infected with the Ba71V strain (GenBank: NC_001659.2), with the exception of p72 which was amplified from cells infected with ASFV E70 (GenBank: AY578692.1). .. PCR products and pcDNA vector were digested with the corresponding restriction enzymes and ligated with T4 ligase (Roche).

    Sedimentation:

    Article Title: N-myc Modulates Expression of p73 in Neuroblastoma
    Article Snippet: Isolated chromosomes were stained with two fluorochromes, Hoechst 33258 and chromomycin A3 (Calbiochem, La Jolla, CA), and allowed to stand for 48 hours using a combination of slow centrifugation and sedimentation. .. Chromosome 1 genomic DNA was digested first with Not I and ligated to the Not I and Eco RV digested plasmid vector Bluescript SK+ (Strategene, La Jolla, CA) with T4 ligase (Roche Molecular Biochemicals, Indianapolis, IN).

    Generated:

    Article Title: The ClgR Protein Regulates Transcription of the clpP Operon in Bifidobacterium breve UCC 2003
    Article Snippet: The resultant 566-bp PCR fragment was digested with BamHI and HindIII and ligated into similarly restricted pQE30, using the T4 DNA ligase enzyme (Roche, Sussex, United Kingdom), to generate plasmid pQE-ClgR, which was introduced into E. coli M15 (QIAGEN, United Kingdom) as described by Sambrook et al. ( ). .. Plasmid pNZ272 , which contains a promoterless gusA gene system, was used as a reporter system.

    DNA Sequencing:

    Article Title: DNA-Protein Vaccination Strategy Does Not Protect from Challenge with African Swine Fever Virus Armenia 2007 Strain
    Article Snippet: PCR products and pcDNA vector were digested with the corresponding restriction enzymes and ligated with T4 ligase (Roche). .. PCR products and pcDNA vector were digested with the corresponding restriction enzymes and ligated with T4 ligase (Roche).

    Article Title: Insights into Lantibiotic Immunity Provided by Bioengineering of LtnI
    Article Snippet: DNA ligations were executed according to established procedures using T4 ligase supplied by Roche Diagnostics. .. DNA ligations were executed according to established procedures using T4 ligase supplied by Roche Diagnostics.

    Sequencing:

    Article Title: Negative Feedback and Transcriptional Overshooting in a Regulatory Network for Horizontal Gene Transfer
    Article Snippet: Strains used were Escherichia coli C41 (ompT hsdS B (rB − mB − ) gal dcm (DE3)), E. coli Bw27783 (lacI q rrnB3 ΔlacZ4787 hsdR514 Δ(araBAD )567 Δ(rhaBAD )568 Δ(araFGH ) Φ(ΔaraEp PCP8 -araE )) and E. coli JM109 (recA1, endA1, gyrA96, thi, hsdR17, supE44, relA1, Δ(lac-proAB)/F′ [traD36, proAB+ , lacIq , lacZΔM15] ) Primer oligonucleotides (Supporting ) were designed to flank each R388 intergenic region longer than 30 bp, according to R388 genomic sequence (Genbank accession number BR000038) and purchased from Sigma. .. Digested and purified fragments were ligated into pUA66 plasmid DNA using T4 ligase (Roche) with overnight incubatio n at 16°C.

    Article Title: Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines
    Article Snippet: To generate LCmCherry V2, the mCherry sequence was obtained from FU-mCherry-w (derived from FUGW) [ ] and then digested with Bsi WI and Sac II restriction enzymes (New England Biolabs, Ipswich, MA, USA). .. The resulting 0.7 kb amplicon was then purified from the agarose gel (Qiagen DNA extraction kit, Hilden, Germany) and subsequently ligated (T4 ligase, Roche, Basel, Switzerland) into the LentiCRISPR Puro V2 at the site of the discarded puromycin fragment (1.3 kb).

    Article Title: Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions
    Article Snippet: All integrin-GFP constructs were made using three-segment (A, B, C) overlap PCR with wild-type human αV cDNA and either pEGFP-N1 (Clontech, for ɑV-GFP–less-constrained) or moxGFP (for ɑV-GFP–constrained) as sources for GFP cDNA. .. After the three segments had been made and joined together using PCR (Accuprime Pfx, high-fidelity polymerase; Thermo Fisher), the complete A–C sequence and the wild-type ɑV-pcDNA3.1 plasmid were cut with restriction enzymes (New England Biolabs) and ligated together with T4 ligase (Roche) after dephosphorylation (rAPID alkaline phosphatase; Roche) and purification (Qiagen) of the linearized plasmid. .. The overall plasmid integrities were verified with size matching of multisite single-restriction enzyme digestion compared with virtual digest patterns (Serial Cloner), and the inserts were verified by full sequencing.

    Article Title: Production, active staining and gas chromatography assay analysis of recombinant aminopeptidase P from Lactococcus lactis ssp. lactis DSM 20481
    Article Snippet: Paragraph title: Cloning, construction of expression vectors and sequencing of pepP ... Ligations were performed according to manufacturer’s (Promega) protocols using T4-ligase (Roche).

    Article Title: Distinct Amino Termini of Two Human HCS Isoforms Influence Biotin Acceptor Substrate Recognition
    Article Snippet: The coding sequence for 58-HCS was amplified from a pGEX construct provided by Dr. Roy Gravel. .. The FL-HCS cDNA sequence (GenBankTM accession number ) was purchased from OriGene Technologies, Inc. After digestion of the PCR product with Esp3I (Fermentas), the resulting two coding sequence fragments were ligated into the linearized pSUMO vector using T4 ligase (Roche Applied Science), and the ligation mixtures were transformed into the E. coli top10 strain. .. The plasmids encoding His6 -SUMO-FL-HCS and His6 -SUMO-58-HCS were transformed into E. coli RosettaTM (DE3).

    Article Title: Crystal Structure of the NS3 Protease-Helicase from Dengue Virus
    Article Snippet: The fragment was digested with XhoI and cloned into a modified pET32b plasmid using T4 ligase (Roche), where the S tag and enterokinase cleavage sequence are absent. .. The PCR fragment was digested with XhoI and cloned into a modified pET32b plasmid using T4 ligase (Roche).

    Article Title: Isolation and Transcription Profiling of Low-O2 Stress-Associated cDNA Clones from the Flooding-stress-tolerant FR13A Rice Genotype
    Article Snippet: First-strand cDNA synthesis was initiated from the enriched mRNA of the tester and driver tissues using different poly (dT)-linker primers (with the sequence 5′-TATAGATCTGCGGCCGCAAGCTTTTTTTTT-3′ for the tester tissues and 5′-GTAATACGACTCACTATAGGGTTTTTTTTT-3′ for the driver tissues) and M-MLV Reverse Transcriptase (Promega Inc.). .. First strand cDNA was purified using Qiaquick columns (Qiagen) and specific oligonucleotides (with sequences 5′-GCTAGCATATGGGCCCGAATTCC-3′ for the tester and 5′-CCCTTTAGTGAGGGTTAATTTC-3′ for the driver) were ligated at the 3′ end of the respective cDNA populations using T4 RNA ligase (Roche).

    Sonication:

    Article Title: Identification of a Novel Streptococcal Gene Cassette Mediating SOS Mutagenesis in Streptococcus uberis
    Article Snippet: Oligonucleotide pairs p26/p27 and p28/p29 with overhangs creating EcoRI and XbaI compatible ends, respectively, were annealed, treated with T4 polynucleotide kinase (MBI Fermentas), and ligated with T4 ligase (Roche) to XbaI-EcoRI cut pBluescript-II SK+ to obtain pBluescript-IR and pBluescript-ctrl, respectively. .. Oligonucleotide pairs p26/p27 and p28/p29 with overhangs creating EcoRI and XbaI compatible ends, respectively, were annealed, treated with T4 polynucleotide kinase (MBI Fermentas), and ligated with T4 ligase (Roche) to XbaI-EcoRI cut pBluescript-II SK+ to obtain pBluescript-IR and pBluescript-ctrl, respectively.

    Binding Assay:

    Article Title: A Fundamental Regulatory Mechanism Operating through OmpR and DNA Topology Controls Expression of Salmonella Pathogenicity Islands SPI-1 and SPI-2
    Article Snippet: SmaI and XbaI digested amplicons were spin column purified, then ligated to pZep or pZec by T4 ligase (Roche). .. The exception was the ompR promoter region which was digested with NotI and XbaI before cloning into similarly digested vector.

    DNA Extraction:

    Article Title: Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines
    Article Snippet: To generate LCmCherry V2, the mCherry sequence was obtained from FU-mCherry-w (derived from FUGW) [ ] and then digested with Bsi WI and Sac II restriction enzymes (New England Biolabs, Ipswich, MA, USA). .. The resulting 0.7 kb amplicon was then purified from the agarose gel (Qiagen DNA extraction kit, Hilden, Germany) and subsequently ligated (T4 ligase, Roche, Basel, Switzerland) into the LentiCRISPR Puro V2 at the site of the discarded puromycin fragment (1.3 kb). .. Secondly, the full length U6 promoter from zebrafish (U6ZF) was amplified by PCR from genomic DNA Danio rerio , using FwU6ZF and RvU6Zf primers.

    Molecular Cloning:

    Article Title: Roles of the Essential Protein FtsA in Cell Growth and Division in Streptococcus pneumoniae
    Article Snippet: Standard protocols for molecular cloning, transformation, and DNA analysis were used ( ). .. Restriction enzymes, DNA polymerase, and T4 ligase were purchased from Roche Diagnostics or New England BioLabs.

    Fluorescence:

    Article Title: Identification of a Novel Streptococcal Gene Cassette Mediating SOS Mutagenesis in Streptococcus uberis
    Article Snippet: The primers p24 and p25 carried HEX (4,7,2′,4′,5′,7′-hexachloro-6-carboxyfluorescein) fluorescence label at their 5′ end. .. Oligonucleotide pairs p26/p27 and p28/p29 with overhangs creating EcoRI and XbaI compatible ends, respectively, were annealed, treated with T4 polynucleotide kinase (MBI Fermentas), and ligated with T4 ligase (Roche) to XbaI-EcoRI cut pBluescript-II SK+ to obtain pBluescript-IR and pBluescript-ctrl, respectively.

    Isolation:

    Article Title: N-myc Modulates Expression of p73 in Neuroblastoma
    Article Snippet: Isolated chromosomes were stained with two fluorochromes, Hoechst 33258 and chromomycin A3 (Calbiochem, La Jolla, CA), and allowed to stand for 48 hours using a combination of slow centrifugation and sedimentation. .. Chromosome 1 genomic DNA was digested first with Not I and ligated to the Not I and Eco RV digested plasmid vector Bluescript SK+ (Strategene, La Jolla, CA) with T4 ligase (Roche Molecular Biochemicals, Indianapolis, IN).

    Article Title: The ClgR Protein Regulates Transcription of the clpP Operon in Bifidobacterium breve UCC 2003
    Article Snippet: The resultant 566-bp PCR fragment was digested with BamHI and HindIII and ligated into similarly restricted pQE30, using the T4 DNA ligase enzyme (Roche, Sussex, United Kingdom), to generate plasmid pQE-ClgR, which was introduced into E. coli M15 (QIAGEN, United Kingdom) as described by Sambrook et al. ( ). .. The resultant PCR amplicons were digested with BglII and PstI and ligated into similarly restricted pNZ272, which was used to transform E. coli M15 (QIAGEN, United Kingdom).

    Article Title: Isolation and Transcription Profiling of Low-O2 Stress-Associated cDNA Clones from the Flooding-stress-tolerant FR13A Rice Genotype
    Article Snippet: For construction of libraries, total RNA from root tissues of driver and tester samples was isolated using guanidine thiocyanate according to . mRNA was enriched from the total RNA population by using the PolyATract mRNA isolation kit (Promega Inc.). .. First strand cDNA was purified using Qiaquick columns (Qiagen) and specific oligonucleotides (with sequences 5′-GCTAGCATATGGGCCCGAATTCC-3′ for the tester and 5′-CCCTTTAGTGAGGGTTAATTTC-3′ for the driver) were ligated at the 3′ end of the respective cDNA populations using T4 RNA ligase (Roche).

    Lysis:

    Article Title: Insights into Lantibiotic Immunity Provided by Bioengineering of LtnI
    Article Snippet: For colony PCR, genomic DNA was accessed through lysis of cells in 10% Igepal CA-630 (Sigma-Aldrich) at 94°C for 10 min. .. DNA ligations were executed according to established procedures using T4 ligase supplied by Roche Diagnostics.

    Electrophoretic Mobility Shift Assay:

    Article Title: Identification of a Novel Streptococcal Gene Cassette Mediating SOS Mutagenesis in Streptococcus uberis
    Article Snippet: Gel shift reactions were incubated at 25°C for 15 min followed by electrophoresis on a 5% polyacrylamide gel with 0.5× TBE (0.0445 M Tris, 0.0455 M borate, 1.25 M EDTA) at room temperature. .. Oligonucleotide pairs p26/p27 and p28/p29 with overhangs creating EcoRI and XbaI compatible ends, respectively, were annealed, treated with T4 polynucleotide kinase (MBI Fermentas), and ligated with T4 ligase (Roche) to XbaI-EcoRI cut pBluescript-II SK+ to obtain pBluescript-IR and pBluescript-ctrl, respectively.

    Purification:

    Article Title: Negative Feedback and Transcriptional Overshooting in a Regulatory Network for Horizontal Gene Transfer
    Article Snippet: PCR amplification was carried out with Vent DNA polymerase (Biolabs) and consisted of 95°C for 10 min, then 28 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s and a final step of 72°C for 5 min. PCR products were digested with XhoI and BamHI at 37°C for 2 h and the products purified using QIAquick Gel Extraction Kit (Qiagen). .. Digested and purified fragments were ligated into pUA66 plasmid DNA using T4 ligase (Roche) with overnight incubatio n at 16°C. .. Transformation was accomplished by electroporation into Bw27783 strain.

    Article Title: A Fundamental Regulatory Mechanism Operating through OmpR and DNA Topology Controls Expression of Salmonella Pathogenicity Islands SPI-1 and SPI-2
    Article Snippet: Gene promoter sequences were PCR amplified using the Phusion DNA polymerase (NEB) and primers listed in . .. SmaI and XbaI digested amplicons were spin column purified, then ligated to pZep or pZec by T4 ligase (Roche). .. The exception was the ompR promoter region which was digested with NotI and XbaI before cloning into similarly digested vector.

    Article Title: Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines
    Article Snippet: To generate LCmCherry V2, the mCherry sequence was obtained from FU-mCherry-w (derived from FUGW) [ ] and then digested with Bsi WI and Sac II restriction enzymes (New England Biolabs, Ipswich, MA, USA). .. The resulting 0.7 kb amplicon was then purified from the agarose gel (Qiagen DNA extraction kit, Hilden, Germany) and subsequently ligated (T4 ligase, Roche, Basel, Switzerland) into the LentiCRISPR Puro V2 at the site of the discarded puromycin fragment (1.3 kb). .. Secondly, the full length U6 promoter from zebrafish (U6ZF) was amplified by PCR from genomic DNA Danio rerio , using FwU6ZF and RvU6Zf primers.

    Article Title: Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions
    Article Snippet: All integrin-GFP constructs were made using three-segment (A, B, C) overlap PCR with wild-type human αV cDNA and either pEGFP-N1 (Clontech, for ɑV-GFP–less-constrained) or moxGFP (for ɑV-GFP–constrained) as sources for GFP cDNA. .. After the three segments had been made and joined together using PCR (Accuprime Pfx, high-fidelity polymerase; Thermo Fisher), the complete A–C sequence and the wild-type ɑV-pcDNA3.1 plasmid were cut with restriction enzymes (New England Biolabs) and ligated together with T4 ligase (Roche) after dephosphorylation (rAPID alkaline phosphatase; Roche) and purification (Qiagen) of the linearized plasmid. .. The overall plasmid integrities were verified with size matching of multisite single-restriction enzyme digestion compared with virtual digest patterns (Serial Cloner), and the inserts were verified by full sequencing.

    Article Title: Production, active staining and gas chromatography assay analysis of recombinant aminopeptidase P from Lactococcus lactis ssp. lactis DSM 20481
    Article Snippet: The purified PCR products and the expression vector [pET20b (+)] were digested with the NdeI and XhoI restriction enzymes and purified as described above. .. Ligations were performed according to manufacturer’s (Promega) protocols using T4-ligase (Roche).

    Article Title: The ClgR Protein Regulates Transcription of the clpP Operon in Bifidobacterium breve UCC 2003
    Article Snippet: The E. coli pQE-30 vector (QIAGEN) was used for overproduction and purification of an N-terminally six-histidine-tagged bifidobacterial ClgR protein (h-ClgR). .. The resultant 566-bp PCR fragment was digested with BamHI and HindIII and ligated into similarly restricted pQE30, using the T4 DNA ligase enzyme (Roche, Sussex, United Kingdom), to generate plasmid pQE-ClgR, which was introduced into E. coli M15 (QIAGEN, United Kingdom) as described by Sambrook et al. ( ).

    Article Title: Differential expression of alphaB-crystallin and Hsp27-1 in anaplastic thyroid carcinomas because of tumor-specific alphaB-crystallin gene (CRYAB) silencing
    Article Snippet: BamH1, EcoR1, Boehringer Mannheim chemoluminescence blotting substrate, T4 deoxyribonucleic acid (DNA) ligase, and TRIzol® reagent were obtained from Roche Diagnostics (Mannheim, Germany). .. BamH1, EcoR1, Boehringer Mannheim chemoluminescence blotting substrate, T4 deoxyribonucleic acid (DNA) ligase, and TRIzol® reagent were obtained from Roche Diagnostics (Mannheim, Germany).

    Article Title: Isolation and Transcription Profiling of Low-O2 Stress-Associated cDNA Clones from the Flooding-stress-tolerant FR13A Rice Genotype
    Article Snippet: The double-stranded mRNA–cDNA hybrids were converted to a single-stranded cDNA population by digestion of mRNA with RNaseH. .. First strand cDNA was purified using Qiaquick columns (Qiagen) and specific oligonucleotides (with sequences 5′-GCTAGCATATGGGCCCGAATTCC-3′ for the tester and 5′-CCCTTTAGTGAGGGTTAATTTC-3′ for the driver) were ligated at the 3′ end of the respective cDNA populations using T4 RNA ligase (Roche). .. The first-strand cDNA of the driver was amplified using excess of biotin-labelled forward primer (corresponding to the respective ligated oligonucleotide) and a reverse primer (corresponding to the respective linker-primer).

    Polymerase Chain Reaction:

    Article Title: DNA-Protein Vaccination Strategy Does Not Protect from Challenge with African Swine Fever Virus Armenia 2007 Strain
    Article Snippet: The fragments were amplified by PCR using the specific primers for each gene from lysate of cells infected with the Ba71V strain (GenBank: NC_001659.2), with the exception of p72 which was amplified from cells infected with ASFV E70 (GenBank: AY578692.1). .. PCR products and pcDNA vector were digested with the corresponding restriction enzymes and ligated with T4 ligase (Roche). .. Products were used to transform E. coli DH5α by heat shock.

    Article Title: Negative Feedback and Transcriptional Overshooting in a Regulatory Network for Horizontal Gene Transfer
    Article Snippet: PCR amplification was carried out with Vent DNA polymerase (Biolabs) and consisted of 95°C for 10 min, then 28 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s and a final step of 72°C for 5 min. PCR products were digested with XhoI and BamHI at 37°C for 2 h and the products purified using QIAquick Gel Extraction Kit (Qiagen). .. Digested and purified fragments were ligated into pUA66 plasmid DNA using T4 ligase (Roche) with overnight incubatio n at 16°C.

    Article Title: A Fundamental Regulatory Mechanism Operating through OmpR and DNA Topology Controls Expression of Salmonella Pathogenicity Islands SPI-1 and SPI-2
    Article Snippet: Gene promoter sequences were PCR amplified using the Phusion DNA polymerase (NEB) and primers listed in . .. SmaI and XbaI digested amplicons were spin column purified, then ligated to pZep or pZec by T4 ligase (Roche).

    Article Title: Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines
    Article Snippet: The resulting 0.7 kb amplicon was then purified from the agarose gel (Qiagen DNA extraction kit, Hilden, Germany) and subsequently ligated (T4 ligase, Roche, Basel, Switzerland) into the LentiCRISPR Puro V2 at the site of the discarded puromycin fragment (1.3 kb). .. The resulting 0.7 kb amplicon was then purified from the agarose gel (Qiagen DNA extraction kit, Hilden, Germany) and subsequently ligated (T4 ligase, Roche, Basel, Switzerland) into the LentiCRISPR Puro V2 at the site of the discarded puromycin fragment (1.3 kb).

    Article Title: Insights into Lantibiotic Immunity Provided by Bioengineering of LtnI
    Article Snippet: For colony PCR, genomic DNA was accessed through lysis of cells in 10% Igepal CA-630 (Sigma-Aldrich) at 94°C for 10 min. .. DNA ligations were executed according to established procedures using T4 ligase supplied by Roche Diagnostics.

    Article Title: Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions
    Article Snippet: All integrin-GFP constructs were made using three-segment (A, B, C) overlap PCR with wild-type human αV cDNA and either pEGFP-N1 (Clontech, for ɑV-GFP–less-constrained) or moxGFP (for ɑV-GFP–constrained) as sources for GFP cDNA. .. After the three segments had been made and joined together using PCR (Accuprime Pfx, high-fidelity polymerase; Thermo Fisher), the complete A–C sequence and the wild-type ɑV-pcDNA3.1 plasmid were cut with restriction enzymes (New England Biolabs) and ligated together with T4 ligase (Roche) after dephosphorylation (rAPID alkaline phosphatase; Roche) and purification (Qiagen) of the linearized plasmid. .. The overall plasmid integrities were verified with size matching of multisite single-restriction enzyme digestion compared with virtual digest patterns (Serial Cloner), and the inserts were verified by full sequencing.

    Article Title: Production, active staining and gas chromatography assay analysis of recombinant aminopeptidase P from Lactococcus lactis ssp. lactis DSM 20481
    Article Snippet: The purified PCR products and the expression vector [pET20b (+)] were digested with the NdeI and XhoI restriction enzymes and purified as described above. .. Ligations were performed according to manufacturer’s (Promega) protocols using T4-ligase (Roche).

    Article Title: Distinct Amino Termini of Two Human HCS Isoforms Influence Biotin Acceptor Substrate Recognition
    Article Snippet: The coding sequence for 58-HCS was amplified from a pGEX construct provided by Dr. Roy Gravel. .. The FL-HCS cDNA sequence (GenBankTM accession number ) was purchased from OriGene Technologies, Inc. After digestion of the PCR product with Esp3I (Fermentas), the resulting two coding sequence fragments were ligated into the linearized pSUMO vector using T4 ligase (Roche Applied Science), and the ligation mixtures were transformed into the E. coli top10 strain. .. The plasmids encoding His6 -SUMO-FL-HCS and His6 -SUMO-58-HCS were transformed into E. coli RosettaTM (DE3).

    Article Title: Influence of IS256 on Genome Variability and Formation of Small-Colony Variants in Staphylococcus aureus
    Article Snippet: In brief, the genomic DNA was digested with XmnI (New England BioLabs, Frankfurt am Main, Germany) or AluI (Roche Applied Science GmbH, Mannheim, Germany). .. After circularization of the fragments using the T4 ligase (Roche Applied Science GmbH, Mannheim, Germany), the insertion sites were detected by PCR analysis using the outgoing primers IS91rev and IS141for, which anneal within the 5′ end of IS 256 ( ). .. Afterwards, the insertion site of the insertion element was detected by Sanger sequencing (SEQLAB Sequence Laboratories Göttingen GmbH, Göttingen, Germany).

    Article Title: Crystal Structure of the NS3 Protease-Helicase from Dengue Virus
    Article Snippet: Den4 NS3hel (residues 177 to 618) was cloned from the full-length NS3 gene by PCR using forward primer 5′-GAAGTGGATGAGGACATT-3′ and reverse primer 5′-GTGGTG CTCGAG TTACTTTCTTCCACTGGCAAA-3′ (the XhoI site is underlined). .. The PCR fragment was digested with XhoI and cloned into a modified pET32b plasmid using T4 ligase (Roche). .. The expression of the Den4 NS3hel protein was done using the same protocol as that described above for scNS2B18 NS3.

    Article Title: Identification of a Novel Streptococcal Gene Cassette Mediating SOS Mutagenesis in Streptococcus uberis
    Article Snippet: Electromobility shift assay (EMSA) reactions (15 μl) were assembled by mixing the PCR-amplified fragment (35 ng) and the His6 -HdiR (0 to 80 ng) in gel shift buffer [20 mM Tris-HCl, pH 8.0, 60 mM KCl, 1 mM dithiothreitol, 10% glycerol, 0.1 mg/ml bovine serum albumin, and 2 μg poly(dI-dC)]. .. Oligonucleotide pairs p26/p27 and p28/p29 with overhangs creating EcoRI and XbaI compatible ends, respectively, were annealed, treated with T4 polynucleotide kinase (MBI Fermentas), and ligated with T4 ligase (Roche) to XbaI-EcoRI cut pBluescript-II SK+ to obtain pBluescript-IR and pBluescript-ctrl, respectively.

    Article Title: The ClgR Protein Regulates Transcription of the clpP Operon in Bifidobacterium breve UCC 2003
    Article Snippet: The clgR gene from B. breve UCC 2003 was amplified using the primers 903-uni and 903-rev, which contain a BamHI and a HindIII restriction site, respectively. .. The resultant 566-bp PCR fragment was digested with BamHI and HindIII and ligated into similarly restricted pQE30, using the T4 DNA ligase enzyme (Roche, Sussex, United Kingdom), to generate plasmid pQE-ClgR, which was introduced into E. coli M15 (QIAGEN, United Kingdom) as described by Sambrook et al. ( ). .. Plasmid pNZ272 , which contains a promoterless gusA gene system, was used as a reporter system.

    Article Title: Genetic and Transcriptional Organization of the clpC Locus in Bifidobacterium breve UCC 2003
    Article Snippet: The PCR was completed by elongation for 10 min at 72°C. .. The resultant 566-bp PCR fragment was digested with BamHI (Roche, Sussex, United Kingdom) and HindIII (Roche, Sussex, United Kingdom) and ligated into similarly restricted pQE30 using the T4 DNA ligase enzyme (Roche) to generate plasmid pQE-ClgR, which was introduced by electrotransformation into E. coli M15 (QIAGEN, United Kingdom) as described by Sambrook and Russell ( ). .. Regions surrounding the clpC homologue in B. animalis subsp. animalis ATCC 25527 were determined by inverse PCR ( ).

    Article Title: Differential expression of alphaB-crystallin and Hsp27-1 in anaplastic thyroid carcinomas because of tumor-specific alphaB-crystallin gene (CRYAB) silencing
    Article Snippet: TITANIUM One-Step RT-polymerase chain reaction (PCR) Kit was obtained from Clontech (Palo Alto, CA, USA). .. BamH1, EcoR1, Boehringer Mannheim chemoluminescence blotting substrate, T4 deoxyribonucleic acid (DNA) ligase, and TRIzol® reagent were obtained from Roche Diagnostics (Mannheim, Germany).

    Article Title: Isolation and Transcription Profiling of Low-O2 Stress-Associated cDNA Clones from the Flooding-stress-tolerant FR13A Rice Genotype
    Article Snippet: First strand cDNA was purified using Qiaquick columns (Qiagen) and specific oligonucleotides (with sequences 5′-GCTAGCATATGGGCCCGAATTCC-3′ for the tester and 5′-CCCTTTAGTGAGGGTTAATTTC-3′ for the driver) were ligated at the 3′ end of the respective cDNA populations using T4 RNA ligase (Roche). .. The first-strand cDNA of the driver was amplified using excess of biotin-labelled forward primer (corresponding to the respective ligated oligonucleotide) and a reverse primer (corresponding to the respective linker-primer).

    Recombinant:

    Article Title: DNA-Protein Vaccination Strategy Does Not Protect from Challenge with African Swine Fever Virus Armenia 2007 Strain
    Article Snippet: Paragraph title: 2.1. ASFV Recombinant Proteins and Plasmid DNAs for Immunization ... PCR products and pcDNA vector were digested with the corresponding restriction enzymes and ligated with T4 ligase (Roche).

    Article Title: Influence of IS256 on Genome Variability and Formation of Small-Colony Variants in Staphylococcus aureus
    Article Snippet: The localizations of IS 256 and recombinant IS 256 elements in different insertion mutants of S. aureus HG001 were determined by inverse PCR as previously described ( ) ( ). .. After circularization of the fragments using the T4 ligase (Roche Applied Science GmbH, Mannheim, Germany), the insertion sites were detected by PCR analysis using the outgoing primers IS91rev and IS141for, which anneal within the 5′ end of IS 256 ( ).

    Labeling:

    Article Title: Ribonucleoprotein Assembly Defects Correlate with Spinal Muscular Atrophy Severity and Preferentially Affect a Subset of Spliceosomal snRNPs
    Article Snippet: After five washes with the same buffer, bound RNAs were recovered from immunoprecipitates by proteinase K treatment, phenol/chloroform extraction and ethanol precipitation. .. 3′-end labeling experiments were carried out in the presence of [32 P] pCp (3000Ci/mmol) and T4 RNA ligase (Roche) following manufacturer's instructions, and unincorporated nucleotides were removed by centrifugation through micro Bio-Spin P-30 columns (Biorad). .. RNAs were analyzed by electrophoresis on 6% polyacrylamide/8M urea gels and autoradiography.

    De-Phosphorylation Assay:

    Article Title: Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions
    Article Snippet: All integrin-GFP constructs were made using three-segment (A, B, C) overlap PCR with wild-type human αV cDNA and either pEGFP-N1 (Clontech, for ɑV-GFP–less-constrained) or moxGFP (for ɑV-GFP–constrained) as sources for GFP cDNA. .. After the three segments had been made and joined together using PCR (Accuprime Pfx, high-fidelity polymerase; Thermo Fisher), the complete A–C sequence and the wild-type ɑV-pcDNA3.1 plasmid were cut with restriction enzymes (New England Biolabs) and ligated together with T4 ligase (Roche) after dephosphorylation (rAPID alkaline phosphatase; Roche) and purification (Qiagen) of the linearized plasmid. .. The overall plasmid integrities were verified with size matching of multisite single-restriction enzyme digestion compared with virtual digest patterns (Serial Cloner), and the inserts were verified by full sequencing.

    Gel Extraction:

    Article Title: Negative Feedback and Transcriptional Overshooting in a Regulatory Network for Horizontal Gene Transfer
    Article Snippet: PCR amplification was carried out with Vent DNA polymerase (Biolabs) and consisted of 95°C for 10 min, then 28 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s and a final step of 72°C for 5 min. PCR products were digested with XhoI and BamHI at 37°C for 2 h and the products purified using QIAquick Gel Extraction Kit (Qiagen). .. Digested and purified fragments were ligated into pUA66 plasmid DNA using T4 ligase (Roche) with overnight incubatio n at 16°C.

    Article Title: Production, active staining and gas chromatography assay analysis of recombinant aminopeptidase P from Lactococcus lactis ssp. lactis DSM 20481
    Article Snippet: The amplified pepP gene PCR products were purified (QIAquick Gel Extraction Kit) after electrophoresis in an agarose gel [1 % (w/v)]. .. Ligations were performed according to manufacturer’s (Promega) protocols using T4-ligase (Roche).

    Concentration Assay:

    Article Title: Genetic and Transcriptional Organization of the clpC Locus in Bifidobacterium breve UCC 2003
    Article Snippet: Each PCR mixture (50 μl) contained 20 mM Tris-HCl, 50 mM KCl, each deoxynucleoside triphosphate at a concentration of 200 μM, 50 pmol of each primer, 1.5 mM MgCl2 , and 1 U of Taq DNA polymerase (Gibco BRL, Paisley, United Kingdom). .. The resultant 566-bp PCR fragment was digested with BamHI (Roche, Sussex, United Kingdom) and HindIII (Roche, Sussex, United Kingdom) and ligated into similarly restricted pQE30 using the T4 DNA ligase enzyme (Roche) to generate plasmid pQE-ClgR, which was introduced by electrotransformation into E. coli M15 (QIAGEN, United Kingdom) as described by Sambrook and Russell ( ).

    Activated Clotting Time Assay:

    Article Title: Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions
    Article Snippet: After the three segments had been made and joined together using PCR (Accuprime Pfx, high-fidelity polymerase; Thermo Fisher), the complete A–C sequence and the wild-type ɑV-pcDNA3.1 plasmid were cut with restriction enzymes (New England Biolabs) and ligated together with T4 ligase (Roche) after dephosphorylation (rAPID alkaline phosphatase; Roche) and purification (Qiagen) of the linearized plasmid. .. After the three segments had been made and joined together using PCR (Accuprime Pfx, high-fidelity polymerase; Thermo Fisher), the complete A–C sequence and the wild-type ɑV-pcDNA3.1 plasmid were cut with restriction enzymes (New England Biolabs) and ligated together with T4 ligase (Roche) after dephosphorylation (rAPID alkaline phosphatase; Roche) and purification (Qiagen) of the linearized plasmid.

    Liquid Chromatography:

    Article Title: Production, active staining and gas chromatography assay analysis of recombinant aminopeptidase P from Lactococcus lactis ssp. lactis DSM 20481
    Article Snippet: Amplification of the pepP genes was carried out as follows: a preliminary denaturation was performed at 95°C for 5 min, followed by 12 cycles of denaturation (15 s at 95°C), annealing (1 min at 52°C for pepP -Lc or at 60°C for pepP -Lb; both reduced by 1°C per cycle) and extension (2 min at 72°C), with a subsequent 35 cycles of denaturation (15 s at 95°C), annealing (1 min at 50°C for pepP -Lc or 55°C for pepP -Lb) and extension (2 min at 72°C), and a final extension was then performed at 72°C for 10 min. .. Ligations were performed according to manufacturer’s (Promega) protocols using T4-ligase (Roche).

    Plasmid Preparation:

    Article Title: DNA-Protein Vaccination Strategy Does Not Protect from Challenge with African Swine Fever Virus Armenia 2007 Strain
    Article Snippet: The fragments were amplified by PCR using the specific primers for each gene from lysate of cells infected with the Ba71V strain (GenBank: NC_001659.2), with the exception of p72 which was amplified from cells infected with ASFV E70 (GenBank: AY578692.1). .. PCR products and pcDNA vector were digested with the corresponding restriction enzymes and ligated with T4 ligase (Roche). .. Products were used to transform E. coli DH5α by heat shock.

    Article Title: Negative Feedback and Transcriptional Overshooting in a Regulatory Network for Horizontal Gene Transfer
    Article Snippet: PCR amplification was carried out with Vent DNA polymerase (Biolabs) and consisted of 95°C for 10 min, then 28 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s and a final step of 72°C for 5 min. PCR products were digested with XhoI and BamHI at 37°C for 2 h and the products purified using QIAquick Gel Extraction Kit (Qiagen). .. Digested and purified fragments were ligated into pUA66 plasmid DNA using T4 ligase (Roche) with overnight incubatio n at 16°C. .. Transformation was accomplished by electroporation into Bw27783 strain.

    Article Title: Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines
    Article Snippet: Paragraph title: 2.1. Plasmid Vector Construction ... The resulting 0.7 kb amplicon was then purified from the agarose gel (Qiagen DNA extraction kit, Hilden, Germany) and subsequently ligated (T4 ligase, Roche, Basel, Switzerland) into the LentiCRISPR Puro V2 at the site of the discarded puromycin fragment (1.3 kb).

    Article Title: Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions
    Article Snippet: All integrin-GFP constructs were made using three-segment (A, B, C) overlap PCR with wild-type human αV cDNA and either pEGFP-N1 (Clontech, for ɑV-GFP–less-constrained) or moxGFP (for ɑV-GFP–constrained) as sources for GFP cDNA. .. After the three segments had been made and joined together using PCR (Accuprime Pfx, high-fidelity polymerase; Thermo Fisher), the complete A–C sequence and the wild-type ɑV-pcDNA3.1 plasmid were cut with restriction enzymes (New England Biolabs) and ligated together with T4 ligase (Roche) after dephosphorylation (rAPID alkaline phosphatase; Roche) and purification (Qiagen) of the linearized plasmid. .. The overall plasmid integrities were verified with size matching of multisite single-restriction enzyme digestion compared with virtual digest patterns (Serial Cloner), and the inserts were verified by full sequencing.

    Article Title: Production, active staining and gas chromatography assay analysis of recombinant aminopeptidase P from Lactococcus lactis ssp. lactis DSM 20481
    Article Snippet: The purified PCR products and the expression vector [pET20b (+)] were digested with the NdeI and XhoI restriction enzymes and purified as described above. .. Ligations were performed according to manufacturer’s (Promega) protocols using T4-ligase (Roche).

    Article Title: Distinct Amino Termini of Two Human HCS Isoforms Influence Biotin Acceptor Substrate Recognition
    Article Snippet: The coding sequence for 58-HCS was amplified from a pGEX construct provided by Dr. Roy Gravel. .. The FL-HCS cDNA sequence (GenBankTM accession number ) was purchased from OriGene Technologies, Inc. After digestion of the PCR product with Esp3I (Fermentas), the resulting two coding sequence fragments were ligated into the linearized pSUMO vector using T4 ligase (Roche Applied Science), and the ligation mixtures were transformed into the E. coli top10 strain. .. The plasmids encoding His6 -SUMO-FL-HCS and His6 -SUMO-58-HCS were transformed into E. coli RosettaTM (DE3).

    Article Title: N-myc Modulates Expression of p73 in Neuroblastoma
    Article Snippet: Two million chromosomes 1 were sorted into siliconized glass tubes. .. Chromosome 1 genomic DNA was digested first with Not I and ligated to the Not I and Eco RV digested plasmid vector Bluescript SK+ (Strategene, La Jolla, CA) with T4 ligase (Roche Molecular Biochemicals, Indianapolis, IN). .. After the first ligation and phenol/chloroform extraction, the DNA was digested with Eco RV and purified with phenol/chloroform extraction-ethanol precipitation.

    Article Title: Human coagulation factor VIII domain-specific recombinant polypeptide expression
    Article Snippet: The pET-28a(+) vector was purchased from Novagen (Madison, WI, USA), and the pGEX-4T-2 vector was obtained from Amersham Biosciences (Uppsala, Sweden). .. Bam HI and Xho I were purchased from Fermentas (Vilnius, Lithuania), and T4 ligase was obtained from Roche (Indianapolis, IN, USA).

    Article Title: Roles of the Essential Protein FtsA in Cell Growth and Division in Streptococcus pneumoniae
    Article Snippet: Paragraph title: Plasmid construction and DNA manipulation. ... Restriction enzymes, DNA polymerase, and T4 ligase were purchased from Roche Diagnostics or New England BioLabs.

    Article Title: Crystal Structure of the NS3 Protease-Helicase from Dengue Virus
    Article Snippet: Den4 NS3hel (residues 177 to 618) was cloned from the full-length NS3 gene by PCR using forward primer 5′-GAAGTGGATGAGGACATT-3′ and reverse primer 5′-GTGGTG CTCGAG TTACTTTCTTCCACTGGCAAA-3′ (the XhoI site is underlined). .. The PCR fragment was digested with XhoI and cloned into a modified pET32b plasmid using T4 ligase (Roche). .. The expression of the Den4 NS3hel protein was done using the same protocol as that described above for scNS2B18 NS3.

    Article Title: The ClgR Protein Regulates Transcription of the clpP Operon in Bifidobacterium breve UCC 2003
    Article Snippet: The clgR gene from B. breve UCC 2003 was amplified using the primers 903-uni and 903-rev, which contain a BamHI and a HindIII restriction site, respectively. .. The resultant 566-bp PCR fragment was digested with BamHI and HindIII and ligated into similarly restricted pQE30, using the T4 DNA ligase enzyme (Roche, Sussex, United Kingdom), to generate plasmid pQE-ClgR, which was introduced into E. coli M15 (QIAGEN, United Kingdom) as described by Sambrook et al. ( ). .. Plasmid pNZ272 , which contains a promoterless gusA gene system, was used as a reporter system.

    Article Title: Genetic and Transcriptional Organization of the clpC Locus in Bifidobacterium breve UCC 2003
    Article Snippet: The PCR was completed by elongation for 10 min at 72°C. .. The resultant 566-bp PCR fragment was digested with BamHI (Roche, Sussex, United Kingdom) and HindIII (Roche, Sussex, United Kingdom) and ligated into similarly restricted pQE30 using the T4 DNA ligase enzyme (Roche) to generate plasmid pQE-ClgR, which was introduced by electrotransformation into E. coli M15 (QIAGEN, United Kingdom) as described by Sambrook and Russell ( ). .. Regions surrounding the clpC homologue in B. animalis subsp. animalis ATCC 25527 were determined by inverse PCR ( ).

    Article Title: Differential expression of alphaB-crystallin and Hsp27-1 in anaplastic thyroid carcinomas because of tumor-specific alphaB-crystallin gene (CRYAB) silencing
    Article Snippet: BamH1, EcoR1, Boehringer Mannheim chemoluminescence blotting substrate, T4 deoxyribonucleic acid (DNA) ligase, and TRIzol® reagent were obtained from Roche Diagnostics (Mannheim, Germany). .. BamH1, EcoR1, Boehringer Mannheim chemoluminescence blotting substrate, T4 deoxyribonucleic acid (DNA) ligase, and TRIzol® reagent were obtained from Roche Diagnostics (Mannheim, Germany).

    Software:

    Article Title: Identification of a Novel Streptococcal Gene Cassette Mediating SOS Mutagenesis in Streptococcus uberis
    Article Snippet: Images were analyzed using the Aida Image Analyzer software v. 4.03 (Raytest GmbH, Straubenhardt, Germany). .. Oligonucleotide pairs p26/p27 and p28/p29 with overhangs creating EcoRI and XbaI compatible ends, respectively, were annealed, treated with T4 polynucleotide kinase (MBI Fermentas), and ligated with T4 ligase (Roche) to XbaI-EcoRI cut pBluescript-II SK+ to obtain pBluescript-IR and pBluescript-ctrl, respectively.

    Article Title: Ribonucleoprotein Assembly Defects Correlate with Spinal Muscular Atrophy Severity and Preferentially Affect a Subset of Spliceosomal snRNPs
    Article Snippet: 3′-end labeling experiments were carried out in the presence of [32 P] pCp (3000Ci/mmol) and T4 RNA ligase (Roche) following manufacturer's instructions, and unincorporated nucleotides were removed by centrifugation through micro Bio-Spin P-30 columns (Biorad). .. RNAs were analyzed by electrophoresis on 6% polyacrylamide/8M urea gels and autoradiography.

    Positron Emission Tomography:

    Article Title: Human coagulation factor VIII domain-specific recombinant polypeptide expression
    Article Snippet: The pET-28a(+) vector was purchased from Novagen (Madison, WI, USA), and the pGEX-4T-2 vector was obtained from Amersham Biosciences (Uppsala, Sweden). .. Bam HI and Xho I were purchased from Fermentas (Vilnius, Lithuania), and T4 ligase was obtained from Roche (Indianapolis, IN, USA).

    Agarose Gel Electrophoresis:

    Article Title: Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines
    Article Snippet: To generate LCmCherry V2, the mCherry sequence was obtained from FU-mCherry-w (derived from FUGW) [ ] and then digested with Bsi WI and Sac II restriction enzymes (New England Biolabs, Ipswich, MA, USA). .. The resulting 0.7 kb amplicon was then purified from the agarose gel (Qiagen DNA extraction kit, Hilden, Germany) and subsequently ligated (T4 ligase, Roche, Basel, Switzerland) into the LentiCRISPR Puro V2 at the site of the discarded puromycin fragment (1.3 kb). .. Secondly, the full length U6 promoter from zebrafish (U6ZF) was amplified by PCR from genomic DNA Danio rerio , using FwU6ZF and RvU6Zf primers.

    Article Title: Production, active staining and gas chromatography assay analysis of recombinant aminopeptidase P from Lactococcus lactis ssp. lactis DSM 20481
    Article Snippet: The amplified pepP gene PCR products were purified (QIAquick Gel Extraction Kit) after electrophoresis in an agarose gel [1 % (w/v)]. .. Ligations were performed according to manufacturer’s (Promega) protocols using T4-ligase (Roche).

    Ethanol Precipitation:

    Article Title: Ribonucleoprotein Assembly Defects Correlate with Spinal Muscular Atrophy Severity and Preferentially Affect a Subset of Spliceosomal snRNPs
    Article Snippet: After five washes with the same buffer, bound RNAs were recovered from immunoprecipitates by proteinase K treatment, phenol/chloroform extraction and ethanol precipitation. .. 3′-end labeling experiments were carried out in the presence of [32 P] pCp (3000Ci/mmol) and T4 RNA ligase (Roche) following manufacturer's instructions, and unincorporated nucleotides were removed by centrifugation through micro Bio-Spin P-30 columns (Biorad).

    Electro Mobility Shift Assay:

    Article Title: Identification of a Novel Streptococcal Gene Cassette Mediating SOS Mutagenesis in Streptococcus uberis
    Article Snippet: Electromobility shift assay (EMSA) reactions (15 μl) were assembled by mixing the PCR-amplified fragment (35 ng) and the His6 -HdiR (0 to 80 ng) in gel shift buffer [20 mM Tris-HCl, pH 8.0, 60 mM KCl, 1 mM dithiothreitol, 10% glycerol, 0.1 mg/ml bovine serum albumin, and 2 μg poly(dI-dC)]. .. Oligonucleotide pairs p26/p27 and p28/p29 with overhangs creating EcoRI and XbaI compatible ends, respectively, were annealed, treated with T4 polynucleotide kinase (MBI Fermentas), and ligated with T4 ligase (Roche) to XbaI-EcoRI cut pBluescript-II SK+ to obtain pBluescript-IR and pBluescript-ctrl, respectively.

    Immunoprecipitation:

    Article Title: Ribonucleoprotein Assembly Defects Correlate with Spinal Muscular Atrophy Severity and Preferentially Affect a Subset of Spliceosomal snRNPs
    Article Snippet: Paragraph title: Immunoprecipitation experiments ... 3′-end labeling experiments were carried out in the presence of [32 P] pCp (3000Ci/mmol) and T4 RNA ligase (Roche) following manufacturer's instructions, and unincorporated nucleotides were removed by centrifugation through micro Bio-Spin P-30 columns (Biorad).

    Two Tailed Test:

    Article Title: Ribonucleoprotein Assembly Defects Correlate with Spinal Muscular Atrophy Severity and Preferentially Affect a Subset of Spliceosomal snRNPs
    Article Snippet: 3′-end labeling experiments were carried out in the presence of [32 P] pCp (3000Ci/mmol) and T4 RNA ligase (Roche) following manufacturer's instructions, and unincorporated nucleotides were removed by centrifugation through micro Bio-Spin P-30 columns (Biorad). .. Immunoprecipitated snRNAs were quantified using a STORM 860 Phosphorimager (Molecular Dynamics) and the ImageQuant version 4.2 software.

    CTG Assay:

    Article Title: Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions
    Article Snippet: After the three segments had been made and joined together using PCR (Accuprime Pfx, high-fidelity polymerase; Thermo Fisher), the complete A–C sequence and the wild-type ɑV-pcDNA3.1 plasmid were cut with restriction enzymes (New England Biolabs) and ligated together with T4 ligase (Roche) after dephosphorylation (rAPID alkaline phosphatase; Roche) and purification (Qiagen) of the linearized plasmid. .. After the three segments had been made and joined together using PCR (Accuprime Pfx, high-fidelity polymerase; Thermo Fisher), the complete A–C sequence and the wild-type ɑV-pcDNA3.1 plasmid were cut with restriction enzymes (New England Biolabs) and ligated together with T4 ligase (Roche) after dephosphorylation (rAPID alkaline phosphatase; Roche) and purification (Qiagen) of the linearized plasmid.

    Staining:

    Article Title: N-myc Modulates Expression of p73 in Neuroblastoma
    Article Snippet: Isolated chromosomes were stained with two fluorochromes, Hoechst 33258 and chromomycin A3 (Calbiochem, La Jolla, CA), and allowed to stand for 48 hours using a combination of slow centrifugation and sedimentation. .. Chromosome 1 genomic DNA was digested first with Not I and ligated to the Not I and Eco RV digested plasmid vector Bluescript SK+ (Strategene, La Jolla, CA) with T4 ligase (Roche Molecular Biochemicals, Indianapolis, IN).

    Article Title: Identification of a Novel Streptococcal Gene Cassette Mediating SOS Mutagenesis in Streptococcus uberis
    Article Snippet: Oligonucleotide pairs p26/p27 and p28/p29 with overhangs creating EcoRI and XbaI compatible ends, respectively, were annealed, treated with T4 polynucleotide kinase (MBI Fermentas), and ligated with T4 ligase (Roche) to XbaI-EcoRI cut pBluescript-II SK+ to obtain pBluescript-IR and pBluescript-ctrl, respectively. .. Oligonucleotide pairs p26/p27 and p28/p29 with overhangs creating EcoRI and XbaI compatible ends, respectively, were annealed, treated with T4 polynucleotide kinase (MBI Fermentas), and ligated with T4 ligase (Roche) to XbaI-EcoRI cut pBluescript-II SK+ to obtain pBluescript-IR and pBluescript-ctrl, respectively.

    Fluorescence In Situ Hybridization:

    Article Title: Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines
    Article Snippet: The expression vector LentiCRISPR-Cas9-2A-mCherryU6ZF (LcU6ZF, hereafter) created for fish cell lines was based on the mammalian LentiCRISPR Puro V2 from Feng Zhang´s lab, (addgene plasmid #52961) [ ] which was modified in two steps, as follows. .. The resulting 0.7 kb amplicon was then purified from the agarose gel (Qiagen DNA extraction kit, Hilden, Germany) and subsequently ligated (T4 ligase, Roche, Basel, Switzerland) into the LentiCRISPR Puro V2 at the site of the discarded puromycin fragment (1.3 kb).

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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: 99/100, based on 0 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:

    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:

    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:

    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:

    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: T4 DNA ligase (Roche) had a final concentration of 40 units/ml in a 0.5× TBE buffer (pH 8) that was modified as follows.

    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: T4 DNA ligase (Roche) had a final concentration of 40 units/ml in a 0.5× TBE buffer (pH 8) that was modified as follows.

    Techniques: Standard Deviation

    BER analysis of mitochondrial extracts. BER analysis was carried out in duplicate using two independently prepared mitochondrial extracts from HeLa (A) and U2OS (B) cell lines. The increased level of the upper band after T4 DNA ligase treatment relative to the untreated samples indicates the presence of unligated DNA repair intermediates (rep. int.) in the sample. (C) BER analysis of mitochondrial extracts from mouse brain as per panels A and B. The bar charts show the relative intensity of the upper band (fully repaired 24 nucleotides) to the lower bands (repair intermediates) in each lane. The error bars show standard deviation of the mean. Absence of detectable repair products in G:C control DNA substrate indicates site specific repair of uracil in U:G DNA substrate.

    Journal: DNA repair

    Article Title: Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair

    doi: 10.1016/j.dnarep.2014.01.015

    Figure Lengend Snippet: BER analysis of mitochondrial extracts. BER analysis was carried out in duplicate using two independently prepared mitochondrial extracts from HeLa (A) and U2OS (B) cell lines. The increased level of the upper band after T4 DNA ligase treatment relative to the untreated samples indicates the presence of unligated DNA repair intermediates (rep. int.) in the sample. (C) BER analysis of mitochondrial extracts from mouse brain as per panels A and B. The bar charts show the relative intensity of the upper band (fully repaired 24 nucleotides) to the lower bands (repair intermediates) in each lane. The error bars show standard deviation of the mean. Absence of detectable repair products in G:C control DNA substrate indicates site specific repair of uracil in U:G DNA substrate.

    Article Snippet: DNA was resuspended in 10 mM Tris–HCl pH 8.5 and divided into two aliquots; one was incubated with 1 μl T4 DNA ligase (Roche) at 37 °C for 60 min and one was left untreated.

    Techniques: Standard Deviation