AAT Bioquest

What are the general principles of plasmid isolation?

Posted January 25, 2023


Plasmid isolation is an essential step in a number of biological procedures including DNA sequencing, gene therapy, cloning, in vitro translation, and transfection among others. The isolation of high-purity plasmid DNA is crucial in all of these applications. This can be a challenging process as plasmids are highly resistant to permanent denaturation. Plasmid isolation is achieved over 4 main steps that ensure the level of purity required:

  1. Lysis and denaturation – Using a strong base combined with a detergent provides denaturating conditions that are strong enough to weaken the resilient bacterial cell wall and break the hydrogen bonds between the two strands of DNA helices. Cell lysis must be performed quickly in order to avoid denaturing the plasmid irreversibly.
  2. Neutralization – In the neutralization step, the complementary DNA strands reanneal and the proteins are precipitated. The supercoiled structures that hold the 2 strands of the helix together during denaturation allow the plasmids to renature.
  3. Centrifugation – During centrifugation, plasmid DNA is separated from chromosomal DNA and large aggregates of precipitated proteins.
  4. Additional purification step – In this last step, plasmids undergo additional purification either by adsorption to a resin or by organic extraction. 
Additional resources

Complete sequence and comparative genomic analysis of eight native Pseudomonas syringae plasmids belonging to the pPT23A family

Sanger Sequencing

MagaDye™ 4 Color Sanger Sequencing Terminator Kit