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 cohesiveended or blunt-ended termini. RBC Rapid DNA Ligation Kit is designed for efficient ligation of DNA inserts into plasmid vectors in just 5 minutes.
Unit Definition
One unit of enzyme catalyzes the conversion of 1 nanomole of [32PPi] into Norit-adsorbable form in 20 mins 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 5-10 μl mix in water or TE buffer of digested vector DNA (50-400 ng) and foreign DNA to be inserted.
Add the following components to the same tube: i 10X Ligation Buffer A 2 μl ii 10X Ligation Buffer B 2 μl iii T4 DNA Ligase 1 μl iv Nuclease-Free Water to final volume of 20ul
Vortex the tube and spin down in microcentrifuge for 3-5 secs. 6. Incubate the mixture for 5 -20 mins at 22°C. 7. Inactivate T4 DNA Ligase by heating the reaction mixture at 65°C for 10 mins. Use the mixture for transformation.
Joining double-stranded DNA with sticky or blunt termini.
Joining of oligonucleotide linkers to blunt-ended DNA
Repairing nicks in duplex DNA, RNA or DNA-RNA hybrids.
Storage Conditions
RBC Rapid Ligation Kit should be stored immediately upon receipt at -20°C in a constant temperature freezer. RBC Rapid Ligation Kit can be stored for up to 12 months without showing any deduction in performance and quality with proper storage.
Note:
T4 DNA Ligase is strongly inhibited by NaCl or KCl if the concentration exceeds 0.2M.
10X Ligation Buffer B greatly increases the rate of ligation of blunt-ended DNA.
The inactivation of T4 DNA Ligase by heating at 65°C for 10 minutes is recommended as a standard procedure prior to transformation of cells with DNA. In some cases, this simple step can increase the number of transformants by two orders of magnitude.
Transformation efficiency is increased if DNA is extracted prior to transformation, Use equal or higher (up to 3-fold) molar concentration of insert DNA termini over vector DNA. If the yield of ligation product is insufficient, prolong the reaction time (overnight), Ligation reactions performed at lower temperatures require longer incubation times.
