Centrifuge the overnight bacterial culture to pellet the cells. Remove the supernatant and resuspend the cell pellet in TE buffer.
Add a denaturing solution to raise the pH and lyse the cells, releasing their contents into solution.
Add renaturing solution to lower the pH, causing proteins and genomic DNA to precipitate while leaving plasmid DNA in suspension.
Centrifuge the sample to pellet proteins and genomic DNA. Transfer the supernatant containing plasmid DNA to a new tube.
Add ethanol to precipitate the plasmid DNA, then either centrifuge to pellet it or transfer the sample to a DNA-binding column.
Wash the pellet or column with 70% ethanol to remove salts and contaminants. Elute or resuspend the DNA using water or neutral buffer.
Optionally enhance plasmid purity by adding RNase A after lysis or performing a phenol-chloroform extraction to remove remaining contaminants.
Measure the concentration and purity of the final plasmid DNA solution before proceeding to downstream applications or assays.
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