A Peculiar Event In Tranformation

bipin k. dalmia dalmiabk at phibred.com
Wed Jun 18 16:26:56 EST 1997


On Tue, 17 Jun 1997 12:39:31 -0400, Randy Willis
<willis at gandalf.psf.sickkids.on.ca> wrote:

>I think what you may be experiencing is normal for bacterial colonies
>which are stored on plates.  As a colony ages, even at 4C, there is less
>stress on the colony to maintain it's plasmid.  This may be due to
>reduced metabolic requirements at 4C or may be due to diffusable
>antibiotic resistance gene products (ie b-lactamase) leaking into the
>medium and eating up the antibiotic.  As the stress is reduced, the
>cells can live free of their plasmid and will "spit it out" with time. 
>This has been manifested in our lab by the finding that, if a plate is
>allowed to sit for too long (a month or so) at 4C, the colonies on the
>plate will no longer grow under selective conditions.  I almost lost a
>couple of constructs this way and could only save them by harvesting all
>of the colonies from the plate and performing a miniprep...whew!
>
>In our lab, whenever we are preparing to do a growth/expression of our
>proteins of interest, we have gotten into the habit of performing a
>fresh transformation reaction.  This seems to give us more reproduceable
>expression results and we handwave that this is due to a higher copy
>number of plasmids when the cultures start.  During the liquid growth
>phase, we also hedge our bets by centrifuging the cells from the media
>when inoculating a larger culture from a smaller overnight.  This limits
>the amount of b-lactamase that is transfered from the overnight to the
>new culture and seems to increase our yields.

good explanation, but the presumption is that the selection was
ampicillin based. kanamycin is not degraded and also carbenicillin
which is a replacement for ampicillin (although much more expensive). 

in any case, restreak your plates every 2 weeks and for longer term
storage, store the plasmid and not glycerol stocks and retransform
when you need them.

bip



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