Loss of plasmid in liquid culture

Martin Kennedy mkennedy at chmeds.ac.nz
Sat Mar 19 04:34:06 EST 1994


In article <94075.051728RROHAN at UMAB.BITNET>, <RROHAN at UMAB.BITNET> writes:
> 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
> Rich Rohan, University of Maryland School of Medicine  RROHAN at UMAB.UMD.EDU

Have you streaked the transformed bugs out onto an Amp plate before doing the
large scale prep?  I know it sounds old fashioned, but if you are growing
direct from a transformed colony, then you are inoculating your culture with a
lot of viable cells that don't contain plasmid.  These will take off and
probably overgrow your poor-yielding plasmid+ strain the minute all the Amp has
been inactivated by the resistant bugs (which doesn't take very long).  The 
sensitive cells have a shorter doubling time because they aren't carrying 
plasmid, and they'll rapidly take over the culture. 

No flame intended, but as a general observation people frequently  blame 
deletions or lethal products for apparent loss of plasmids or inserts, but I 
think there is far more often a trivial reason for these apparent problems.

-- 
Cheers,

Martin

NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
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