DNA ligation

• Set up the following reactions in a PCR tube on ice:

T4 DNA ligase should be added last, molar ratio of vector:insert DNA should be about 3:1

2ul 10x T4 DNA ligase buffer

100-300ng vector DNA

100-300ng insert DNA

water to 20ul

1ul T4 DNA ligase

Make sure that the ligase buffer is completely thawed and mixed by vortexing. It contains NADH as a cofactor for the reaction and this often ppts out on initial thawing.

• Mix reactants by pipetting up and down

• Incubate at room temperature for 10 mins (sticky end cloning). Increase time to 2hrs for blunt end cloning.

• Cool on ice and transform 1-5ul into 50ul competent cells