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AAT Bioquest

How do I remove genomic DNA contamination from isolated plasmid DNA?

Posted September 25, 2020


Answer

The separation of plasmid DNA from genomic DNA contaminates (e.g. chromosomal DNA and cellular RNA) requires a milder alkaline treatment. First, bacterial cells are cultivated and resuspended in an isotonic solution (e.g. TE buffer containing). Then an SDS-alkaline solution is used to lyse the cell membrane and denature both genomic, plasmid DNA and any proteins in the solution. Next the sample is neutralized using a potassium acetate solution. This separates the plasmid DNA from the genomic DNA by causing the genomic DNA to precipitate out of the solution. Finally, the genomic DNA can be spun down into pellet form via centrifugation, while the plasmid DNA, which remains in the supernatant, can be purified using a phenol-chloroform mixture.

Additional resources

A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples

DNA and RNA Quantitation