Fast Ligation Kit

Description

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. Fast DNA Ligation Kit is designed for efficient ligation of DNA inserts into plasmid vectors in just 10 minutes.

Applications

Suitable for all common ligation reactions including cohesive and blunt end ligations.

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

Storage

The kit is stable for 12 months at -20oC.

Features

Fast - 10 minute protocol for both cohesive and blunt end ligations.

Protocol

1. Add de-ionized water or Tris-EDTA (TE) buffer to 50ng of vector and 15 ng insert (3 fold excess of insert is recommended), to a final volume to 9ul.
2. Add 10ul of 2X reaction buffer and mix by inverting tube several times.
3. Add 1ul of Fast DNA Ligase and mix thoroughly by inverting tube several times. Briefly centrifuge to bring the contents to the bottom of the tube.
4. Incubate the ligation mixture at room temperature for 10 minutes1.
5. If transforming competent cells (HIT DH5 alpha Catalog Number RH617) store ligation reaction on ice until transformation. Alternatively the reaction can be stored at -20°C for extended periods of time.

Notes:

1. Increasing the incubation time from 10 to 20 minutes has been shown to increase ligation efficiency for certain DNA fragments.
2. Inactivation of ligase by heating is not recommended, as it may reduce transformation efficiency.
3. Avoid repeated freeze/thaw cycles for maximum ligase efficiency.