Using Alkaline Lysis Method for Recombine Plasmid DNA Extraction and PCR Verification

Objective:To obtain high-quality plasmid through screening for further cloning and sequencing of work to lay the foundation. Method: Plasmid DNA was extracted from recombine E.coli. By using the conventional alkaline lysis method. The isolated plasmid DNA was quantified by nucleic acid detector, and used the previous primers as same as DDRT-PCR for PCR verification, then evaluated by the electrophoresis of plasmid DNA in an agarose gel. Result: The use of conventional alkaline lysis method for extracting plasmid DNA by phenol and chloroform extraction can effectively remove protein impurities. Using sterile water which containing RNase inhibitor for dissolving plasmid DNA is the best method, and the plasmid DNA has very high purity and without RNA pollution. The OD260/OD280 ratio is between 1.8 and 2.0, and the OD260/OD230 ratio is bigger than 2.0. PCR verification is expected. Conclusion: The purity and concentration of recombinant plasmid is higher by this method, and the PCR verification is expected, so it can meet the requirements of molecular biology experiments.