Dephosphorylation experiments on enzyme section breaks

Summary

In the modification of DNA fragments, the dephosphorylation reaction is an important element, which is catalyzed by alkaline phosphatase, an enzyme that removes the 5' phosphate group from linear DNA.

Operation method

basic program

Materials and Instruments

DNA fragments
Alkaline phosphatase Phenol Chloroform EDTA SDS TE buffer Electrophoresis buffer NH4AC
Centrifuge Thermostat Vacuum Dryer Liquid Extractor

Move

1. Add to the DNA restriction endonuclease fragments obtained in experiment IV.
(1) 10 × alkaline phosphatase buffer 10 μl
(2) Alkaline phosphatase (0.01 u/pmol ends) 1-2 ul
(3) _ddH2O to 100 ul
(4) For 5' prominence then warm bath at 37°C for 30 min, then add 1 ul CIAP (0.01 u/pmol ends) and warm bath at 37°C for 30 min.
(5) For flat ends or 3' protruding ends, a warm bath at 37°C for 15 min and 56°C for 15 min, then 1 ul CIAP (0.01 u/pmol ends) was added, and the bath was warmed at 37°C for 15 min. 56°C for 15 min.
2. At the end of the warm bath, EDTA (pH 8.0) was added to a final concentration of 10 mM, respectively, and heated at 65°C for 20 min to inactivate alkaline phosphatase.
3. Extract once with phenol and once with chloroform.
4. Add 1/2 volume of _NH4AC, mix thoroughly, then add 2 volumes of ethanol, leave at -20°C for 2 hr (or -70°C for 30 min), centrifuge at 12,000 g for 10 min, and precipitate DNA.
5. Wash the precipitate once with cold 70% ethanol.
6. Dissolve in TE (final concentration 100 μg/μl) and store at -20°C.

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