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- 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.
* The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.
||20 μl REACTION
|T4 DNA Ligase Buffer (10X)*
|Vector DNA (4 kb)
||50 ng (0.020 pmol)
|Insert DNA (1 kb)
||37.5 ng (0.060 pmol)
||to 20 μl
|T4 DNA Ligase
- 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.