T4 dna ligase for guide seq

Manufactured by Qiagen

T4 DNA ligase is an enzyme used in molecular biology applications, such as DNA cloning and sequencing. It catalyzes the formation of phosphodiester bonds between adjacent 3'-hydroxyl and 5'-phosphate termini in double-stranded or single-stranded DNA. This enzyme is commonly used in GUIDE-seq, a method for genome-wide unbiased identification of DNA double-strand break sites.

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T4 DNA ligase (33 citations) T4 ligase (4 citations)

3 protocols using t4 dna ligase for guide seq

CRISPR-Cas9 Genome Editing Workflow

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Restriction enzymes, T4 ligase, Dulbecco’s modified Eagle Medium DMEM (Gibco), fetal bovine serum (Gibco), Turbofect, TranscriptAid T7 High Yield Transcription Kit, Qubit dsDNA HS Assay Kit, Taq DNA polymerase (recombinant), Platinum Taq DNA polymerase, 0.45 µm sterile filters and penicillin/streptomycin were purchased from Thermo Fischer Scientific, protease inhibitor cocktail was purchased from Roche Diagnostics. DNA oligonucleotides, trimethoprim (TMP), chloroquine, polybrene, puromycin, calcium-phosphate and GenElute HP Plasmid Miniprep kit were acquired from Sigma-Aldrich. ZymoPure Plasmid Midiprep kit and RNA Clean & Concentrator kit were purchased from Zymo Research. NEBuilder HiFi DNA Assembly Master Mix and Q5 High-Fidelity DNA Polymerase were obtained from New England Biolabs Inc. NucleoSpin Gel and PCR Clean-up kit was purchased from Macherey-Nagel. Two millimeter electroporation cuvettes was acquired from Cell Projects Ltd, SF Cell Line 4D-Nucleofector X Kit S were purchased from Lonza, Bioruptor 0.5 ml Microtubes for DNA Shearing from Diagenode. Agencourt AMPure XP beads were purchased from Beckman Coulter. T4 DNA ligase (for GUIDE-seq) and end-repair mix were acquired from Enzymatics. KAPA universal qPCR Master Mix was purchased from KAPA Biosystems.

Kulcsár P.I., Tálas A., Ligeti Z., Tóth E., Rakvács Z., Bartos Z., Krausz S.L., Welker Á., Végi V.L., Huszár K, & Welker E. (2023). A cleavage rule for selection of increased-fidelity SpCas9 variants with high efficiency and no detectable off-targets. Nature Communications, 14, 5746.

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Molecular Cloning and Transfection Protocol

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Restriction enzymes, T4 ligase, Dulbecco’s modified Eagle Medium DMEM (Gibco), fetal bovine serum (Gibco), Turbofect, Qubit dsDNA HS Assay Kit, Taq DNA polymerase (recombinant), TranscriptAid T7 High Yield Transcription Kit, Platinum Taq DNA polymerase, 0.45 µm sterile filters and penicillin/streptomycin were purchased from Thermo Fischer Scientific. DNA oligonucleotides, trimethoprim (TMP) and GenElute HP Plasmid Miniprep kit were acquired from Sigma–Aldrich. ZymoPure Plasmid Midiprep, RNA Clean & Concentrator kit and Maxiprep kits were purchased from Zymo Research. NEBuilder HiFi DNA Assembly Master Mix and Q5 High-Fidelity DNA Polymerase were obtained from New England Biolabs Inc. NucleoSpin Gel and PCR Clean-up kit was purchased from Macherey-Nagel. SF Cell Line 4D-Nucleofector X Kit S were purchased from Lonza, Bioruptor 0.5 ml Microtubes for DNA Shearing from Diagenode. Agencourt AMPure XP beads were purchased from Beckman Coulter. T4 DNA ligase (for GUIDE-seq) and end-repair mix were acquired from Enzymatics. KAPA universal qPCR Master Mix was purchased from KAPA Biosystems.

Kulcsár P.I., Tálas A., Ligeti Z., Krausz S.L, & Welker E. (2022). SuperFi-Cas9 exhibits remarkable fidelity but severely reduced activity yet works effectively with ABE8e. Nature Communications, 13, 6858.

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Molecular Biology Reagents and Protocols

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Restriction enzymes, T4 ligase, Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640 medium, fetal bovine serum (FBS), Turbofect, Geltrex, StemPro Accutase, Qubit dsDNA HS Assay Kit, Platinum Taq DNA polymerase, and penicillin/streptomycin were purchased from Thermo Fischer Scientific Inc SF and SE Cell Line 4D-Nucleofector X Kit S were purchased from Lonza. Bioruptor 0.5 ml microtubes for DNA Shearing were from Diagenode. Agencourt AMPure XP beads were purchased from Beckman Coulter. T4 DNA ligase (for GUIDE-seq) and end-repair mix were acquired from Enzymatics. KAPA Universal qPCR Master Mix was purchased from KAPA Biosystems. DNA oligonucleotides and the GenElute HP Plasmid Miniprep and Midiprep kit used for plasmid purifications were acquired from Sigma-Aldrich. Q5 High-Fidelity DNA Polymerase, NEB5-alpha competent cells, and HiFi Assembly Master Mix were purchased from New England Biolabs Inc.

Simon D.A., Tálas A., Kulcsár P.I., Biczók Z., Krausz S.L., Várady G, & Welker E. (2022). PEAR, a flexible fluorescent reporter for the identification and enrichment of successfully prime edited cells. eLife, 11, e69504.

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