Ligation Protocol with T4 DNA Ligase (M0202)
Protocols.io also provides an interactive version of this protocol where you can discover and share optimizations with the research community.
- Set up the following reaction in a microcentrifuge tube on ice.
(T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.) Use NEBioCalculator to calculate molar ratios.
COMPONENT 20 μl REACTION T4 DNA Ligase Buffer (10X)* 2 μl Vector DNA (4 kb) 50 ng (0.020 pmol) Insert DNA (1 kb) 37.5 ng (0.060 pmol) Nuclease-free water to 20 μl T4 DNA Ligase 1 μl
- Gently mix the reaction by pipetting up and down and microfuge briefly.
- For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
- For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).
- Heat inactivate at 65°C for 10 minutes.
- Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.
This tutorial describes the use of the NEBioCalculator web tool to optimize the molar ratio between vector and insert DNA for use in a ligation reaction.