Loss of plasmid in liquid culture

RROHAN at UMAB.BITNET RROHAN at UMAB.BITNET
Wed Mar 16 05:17:28 EST 1994


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



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