Linear plasmid DNA 5'-sticky end dephosphorylation assay

Summary

The 5' end of the plasmid DNA cut by restriction endonuclease has a phosphate group, and if this vector is used to directly transform E. coli, the chance of its own cyclization is very high, which affects the efficiency of ligation and transformation. Therefore, in general, plasmid DNA digested by restriction endonuclease should be dephosphorylated, using alkaline phosphatase (CIAP) to remove the phosphate group at the 5' end.

Operation method

extraction

Materials and Instruments

Plasmids
CIAP EDTA BUFFER Phenol-Chloroform Ethanol TE
Water Bath Extractor

Move

1, plasmid DNA and CIAP and BUFFER 37 ℃ water bath 30min
2, add CIAP and 37 ℃ water bath for 30min.
3、Add 50 mM EDTA to the final concentration of 5 mM 75 ℃ heating 10min inactivation of CIAP
4, cool to room temperature, phenol-chloroform extraction, ethanol precipitation concentration.
5、Drain the solution, add appropriate amount of TE to dissolve.

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