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, 89μl of sterile, distilled H2O. 2) To this solution add 10μl of 10 x OPA+ and 1μl of a 0.1 unit/μl 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℃ for 30 minutes. 4) After incubation is complete, heat inactivate the CIP by incubating the solution at 85℃ 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. |
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