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Materials

Enzymes

Restriction enzymes can be purchased from many manufacturers
Calf intestine alkaline phosphatse

10X restriction enzyme buffer

Restriction enzyme buffers (10X) are usually supplied by the manufacturers with the enzymes. When you would like to carry out a double digestion, you should check if the enzymes are compatiable and which buffer should be used (e.g. in the New England Biolabs catalog 2000/1, p. 204).

10X dephosphorylation buffer

Calf intestine alkaline phosphatase is active in most restriction buffers. Usually it is supplied by the manufacturer with a specific buffer.

Digestions

Mix in 500-µl microfuge tubes:

5 µl 10X restriction enzyme buffer
0.5 µl 100X BSA (; depending on the restriction enzyme)
7.5 µg vector DNA
2.5 µl restriction enzyme 1 (10-20 units/µl)
2.5 µl restriction enzyme 2 (10-20 units/µl)

 
add sterile water to a volume of 50 µl

1. Add the restriction enzymes last.
2. Mix gently by tapping the tube or pipetting the solution up and down.
3. Incubate the reaction mixtures at 37°C for 2-4 h.

Remark:
Some restriction enzymes may display "star activity", a less stringent sequence dependence that results in altered specificity (and more randomly digested vector DNA). Conditions that can lead to star activity include high glycerol concentrations (>5%).

Dephosphorylation (optional)

When the digestion is complete, add to the reaction mixture:

5 µl 10X dephosphorylation buffer
5 µl calf intestine alkaline phosphatase (10 units/µl)
40 µl sterile water

4.  Incubate at 37°C for 30 min.

Purification of digested vector DNA

To separate the digested vecor from the small DNA fragment that was removed by the digestion, the entire sample is purified using preparative agarose gel electrophoresis.

The vector DNA is extracted from the agarose gel using a commercial gel extraction kit.