Note t4 dna ligase is active in pcr and restriction. T4 dna ligase structure reveals a prototypical atpdependent. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3 hydroxyl and 5 phosphate termini. Promega enzyme resource guide, cloning enzymes, br075b. Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and returned immediately to 20 c after use. For blunt ends, use 1 l of t4 dna ligase in a 20 l reaction for 2 hours or 1 l high concentration t4 dna ligase for 10 minutes. Pdf dna unwinding and inhibition of t4 dna ligase by. Mix thoroughly, spin briefly and incubate for 1 hour at 22c. Bacteriophage t4 dna ligase is the first atpdependent ligase enzyme to. Dna ligase is a specific type of enzyme, a ligase, ec 6. Dna ligase i also interacts with replication factor c, the factor that loads the. Multiple dna ligation events are required to join the okazaki fragments generated. Dna unwinding and inhibition of t4 dna ligase by anthracyclines article pdf available in nucleic acids research 169.
Dna ligases are primarily responsible for joining the gaps that form in dna during replication. For blunt ends or single base overhangs, incubate at 16c overnight or room temperature for 2 hours alternatively, high concentration t4 dna ligase can be used in a 10 minute ligation. Learn vocabulary, terms, and more with flashcards, games, and other study tools. T4 dna ligase can be used to join dna fragments with staggered or blunt ends. Nxgen t4 dna ligase high concentration rapid kit lucigen. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex dna and some dnarna hybrids 1. On the basis of an analogous approach to dna 59adenylation using t4 dna ligase chiuman and li 2002 and our own procedure for rna 59adenylation with t4 rna ligase silverman 2004, here we. Dna ligation products product listing application overview neb continues to develop and produce the most extensive commercially available selection of highquality, and performanceoptimized dna ligases and ligase master mixes for your ligation needs. The unique t4 dna ligase buffer optimizes ligation, which can be performed in 5 minutes. Singlestranded nucleic acids are not substrates for thi.
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