Trouble with Glycerol stocks of DH5alpha

John R. Thompson jrt at
Mon Sep 25 05:52:47 EST 1995

A.Cavallaro at (Tony Cavallaro) wrote:

>In out lab lately we have bben trouble growing up DH5alpha cell 
>after they have been glycerol solution frozen at -80 degrees. They grow
>fine in LB-amp (50), but fail to give any DNA after a miniprep procedure.
>THis is unusual since we usually have no trouble with this.
>Also another problem with minipreps, we are also having trouble growing 
>up colonies from plates (both LB agar and Mcconkey). The colonies look 
>good on the plate but do not grow up in liquid culture. We are using 
>DH5alpha again for this.
>Help for this problem would be appreciated.

We've had this problem with this strain in particular.  Run a sample
of the miniprep uncut (i.e. never seen Mg++) .  In our case, this
showed DNA --- add Mg++ and it dissapears.  Nuclease contamination
seems to be the culprit.  We used an additional gene clean step to
clean the DNA up a little more.

John R. Thompson
Merck Research Labs

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