T4 DNA Ligase catalyzes the joining of two strands of DNA between the 5'-phosphate and the 3'-hydroxyl groups of adjacent nucleotides in either a cohesive-ended or blunt-ended termini. RBC Rapid DNA Ligation Kit is designed for efficient ligation of DNA inserts into plasmid vectors in just 5 minutes.


Applications

• Joining double-stranded DNA with cohesive or blunt termini


• Joining of oligonucleotide linkers to blunt-ended DNA


• Repairing nicks in duplex DNA, RNA or DNA-RNA hybrids

20 mM Tris-HCl (pH 7.5), 50 mM KCl, 1mM DTT, 0.1 mM EDTA, and 50 % glycerol Stabilizers


10X Ligation Buffer A

0.4 M Tris-HCl, 0.1 mM MgCl2, 0.1 M DTT, and 5 mM ATP (pH 5.0 at 250C)The performance of this buffer depends on the integrity of the ATP. Store the buffer in small aliquots at -20^°C to minimize degradation of the ATP and DTT.


10X Ligation Buffer B

Contains an enhancer which dramatically increases ligation times for blunt ended DNA.


Unit Definition

One unit of enzyme catalyzes the conversion of 1 nanomole of [32PPi] into Norit-adsorbable form in 20 min at 37^°C(Weiss unit). We recommend using a 1:3 molar ratio of vector : insert DNA when cloning a fragment into a plasmid vector. These ratios will vary with other types of vectors. In a microcentrifuge tube prepare 5-10ul mix in water or TE buffer of digested vector DNA(50-400ng) and foreign DNA to be inserted. Add the following components to the same tube :

• 10X Ligase Buffer A 2 µl


• 10X Ligase Buffer B 2 µl


• T4 DNA Ligase 1 µl


• Nuclease-Free Water to final volume of 20 µl