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DNA ligation

Self ligation

1. Dilution of DNA favors self-ligation.

Linear DNA ( 10-100 ng) 1 ml
10X ligation buffer 10 ml
T4 ligase (5 units) 1 ml
ddH2O 88 ml

Insert into a vector

1a.

Vector DNA ( 20-200 ng) 1 ml
Insert DNA ( 60-600 ng) ( 3 times vector) 1 ml
10X ligation buffer 2 ml
T4 ligase (5 units) 1 ml
ddH2O 15 ml

2. Incubate the ligation mixture at 16 C for 2-3 hours or overnight.

3. Inactivate T4 DNA ligase by heating to 65 C for 10 minutes or use directly for transformation.