Recovering plasmids from frozen cultures

Eric Carstens ecarsten at cgocable.net
Tue Oct 6 20:59:07 EST 1998


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



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