Team:Groningen/Protocols and Protocols/Ligation
Blue Bio Energy
Ligation
Sticky-end ligation of an insert into vector DNA.
ML-I
Ligation
Prepare the following reaction mixture.
Ingredient
Amount
Linear vector DNA
20 – 100 ng
10x T4 DNA ligase buffer
2 μl
T4 DNA ligase
1 U
Water, nuclease-free (= sterile milliQ)
to 20 μl
Total volume
20 μl
Ligation mix.
Incubation
Incubate for 10-60 minutes at 22°C.
Transformation
Use up to 5 μl of the mixture for transformation of 50 μl chemically competent cells or 1-2 μl per 50 μl electrocompetent cells.