De-phosphorylation of DNA

 

De-phosphorylation of DNA

The preferred method of de-phosphorylation uses the buffer system described by Pharmacia for most of their restriction endonucleases.

You will need:

10 x OPA+ Buffer (100mM Tris.acetate, pH 7.5, 100mM magnesium acetate, 500mM potassium acetate)
Calf Intestinal Phosphatase (CIP, Promega)
Sterile, nano-pure water

1) After digestion of DNA with the appropriate restriction endonuclease, phenol/chloroform extract, precipitate and re-dissolve the DNA in, typically, 89ul of sterile, distilled H₂ O.

2) To this solution add 10ul of 10 x OPA+ and 1ul of a 0.1 unit/ul of CIP in 1 x OPA+.

N.B: Do not use more that 0.1 units CIP if it can be avoided as inactivation of CIP by heat is not 100% effective if more than 1 unit is used.

3) Incubate at 37°C for 30 minutes.

4) After incubation is complete, heat inactivate the CIP by incubating the solution at 85°C for 15 minutes.

5) Cool the reaction to room temperature, extract once with phenol:chloroform and once with chloroform.

6) Precipitate DNA with ethanol and recover by centrifugation in a microfuge.

7) Wash the pellet once in 80% ethanol, vacuum dry and resuspend in the appropriate buffer.