Has anyone isolated plasmid DNA from frozen (at -20degrees) E. coli cell cultures (not pellets)? Has that worked and if so, with what yields? What would be the quality of the isolated plasmid DNA if run on a gel? Would most of it be degraded or intact?


1 Answer 1


I have done that and it works without any problems. We harvested the cultures at the optimal timepoint (density) and then froze them to do maxipreps later on. Therefore we simply thawed the culture, made sure it didn't got too warm (meaning we worked on ice) and then simply followed the maxiprep protocol. No problems with yield or quality of the plasmide.

What I will do next time is to spin down the culture and take it up in max. 50 ml of medium before freezing. This way freezing will be faster as well as thawing. I have also done minipreps from stock cultures (glycerol stocks of plasmids in E.coli) which accidentally stayed out of the freezer for two days. Here the yields where not so great (but the density of the culture was also much lower), but the quality after standard miniprep was good and the plasmids worked both for subsequent cloning as well as for retransformation to make new stocks.

  • $\begingroup$ Thanks Chris. I tried it with 25ml cultures and I got great yields! I did not run them on a gel to check the quality of the DNA, but the sequencing results looked good! $\endgroup$ Jul 10, 2014 at 16:43
  • $\begingroup$ Great to hear. In my experience plasmids are incredible stable given how complex this macromolecule is. $\endgroup$
    – Chris
    Jul 10, 2014 at 17:43

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