Loss of plasmid in liquid culture

Michael Benedik bchs1b at Rosie.UH.EDU
Thu Mar 17 15:37:41 EST 1994


In article <94075.051728RROHAN at UMAB.BITNET>, <RROHAN at UMAB.BITNET> writes:
>Dear netters, sorry for any duplication but I believe that my previous posting
>was send to the black hole newsgroup.
>
>I have a clone (pGEM7Zf+ vector with an approx 600 bp insert in HB101) that
>produces colonies on LB/AMP plates.  When we try liquid cultures (LB/AMP) there
>is a very long lag phase and no plasmid DNA can be isolated from the saturated
>cultures.  We have grown this clone in the past although the yield has always
>been poor.  The insert is in-frame with the lacZ indicator in the plasmid (this
>was inadvertant) and could make a fusion product.  My current working
>hypothesis is that this product is lethal.  I don't know what changed in the
>growth conditions that allowed us to growth it before.  My questions:
>1) Is this possible? Has someone had the same experience?
>2) Can I alter the growth conditions and prevent this from happening?
>3) Are there other ways to get around this? (We are currently transferring
>the insert to another vector in the opposite orientation.)
>
>Thanks for any and all comments/suggestions.
>Rich Rohan, University of Maryland School of Medicine  RROHAN at UMAB.UMD.EDU

I have seen this same thing a few times when cloning lethal genes.
Cloning in the opposite orientation will usually solve the problem, however
I would worry that the DNA prep you are working with may have picked
up some mutations to allow survival. so you need to be careful.
What has worked a few times for us with clones that grow on plates but
not liquid, is to spread out a large patch on an LB-AP plate, grow
8-10 hours and scrape all the cells off. Then prepare miniprep the DNA
from those cells and use to subclone.

Good luck

----------------------------------------------------------------------
 Michael Benedik				INTERNET: Benedik at uh.edu
 Dept. of Biochemical & Biophysical Sciences	
 University of Houston				BITNET: Benedik at uhou
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