Recovering plasmids from frozen cultures

luc luc at sgi.celgen.kuleuven.ac.be
Wed Nov 4 12:05:21 EST 1998


we have encountered this problem on a few occasions when we grew a culture
directly from the glycerol stock. it was solved when we first plated the
bacteria on agar.

luc

Eric Carstens wrote:

> We are experiencing a bizzare problem with recovering plasmids from
> frozen bacterial cultures. The original clones (mostly in pBlueScript
> but some are in pBR322 and pUC derivatives, cloned into DH5alpha cells)
> were suspended in 15% glycerol and frozen down at -80C. Recovering
> bacteria from these stocks on amp+ plates yields colonies. However,
> little to no plasmid DNA can be recovered from these bacteria. The
> bacteria are 'amp' resistant but appear to have lost or degrade the
> plasmid.  This is very frustrating, we have been unable to identify the
> problem. We have experienced power failures on the freezer  but we
> believe that the temperature never went above -10C. Does anyone have any
> explanations? In some cases, where we actually were able to recover a
> small amount of plasmid,  we  have been able to recover the clones by
> retransforming cells. In this case, we have used JM109 cells. We have
> not yet retested recovery of these bacteria. Not all of our frozen
> cultures behave in this way, but it seems to occur more frequently with
> relatively large plasmids (10 - 20 kb)
> Any suggestions can be posted or sent to me directly:
>
> Eric Carstens
> Queen's University
> carstens at post.queensu.ca




More information about the Methods mailing list