Ligation Protocol with T3 DNA Ligase (M0317)
- Set up the following reaction in a microcentrifuge tube on ice.
(T3 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 StickTogether™ DNA Ligase Buffer (2X) 10 μl Vector DNA (4 kb) 50 ng (0.020 pmol) Insert DNA (1 kb) 37.5 ng (0.060 pmol) T3 DNA Ligase 1 μl Nuclease-free water to 20 μl
- Gently mix the reaction by pipetting up and down and microfuge briefly.
- Incubate at room temperature (25°C) for 15-30 minutes.
- Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells. Alternatively, store at –20°C.
- Do not heat inactivate – Heat inactivation dramatically reduces transformation efficiency.
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.