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Tuesday, November 18, 2003
Description The preferred method of de-phosphorylation uses the buffer system described by Pharmacia for most of their restriction endonucleases. Procedure 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 H2O. 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 37C for 30 minutes. 4) After incubation is complete, heat inactivate the CIP by incubating the solution at 85C 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. Recipes 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 Supplies Tips (责任编辑：泉水) |
De-phosphorylation of DNA
时间:2005-07-18 00:00来源:Scienceboard.net 作者:admin
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