Insert instability

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
Thu Mar 25 06:33:00 EST 1993


-------------- Jim Gale writes -----------------------------------

Greetings net-folks

I have two questions. First, I have a cDNA clone in pBluescript II which is 
unstable. That is when I grow this clone up from the glycerol stock, 
the majority of plasmid DNA I isolate has lost the insert. These Ecoli cells]
are rec-  but the insert is still being lost (deleted, recombined etc...)
I have re-transfected this plasmid into Stratagenes' SURE cells, which
are deletion mutants for a whole host of Ecoli evils, but the plasmid insert
is still not stable. For all you cloners out there, what tricks have you used
when you find your insert is not stable????

My second question is does anyone know of a vendor for homoginizing 
pestles that fit 1.5 eppendorff tubes. I saw a sample of these at a meeting
but I have lost the name of the supplier

Thanks

Jim Gale
Lovelace Medical foundation
Albuquerque, NM
jim at audrey.lmf.org
----------------------------------------------------------------
Hi Jim!
    This is curious!  What type of events make up your deletions?
Do you have piece of your cDNA clone left or is it totally
missing?  Where did you get this cDNA clone?  From another
vector like lambda or did you PCR it up using one of the variety of
techniques available? I have never really found anything that is not 
stable in plasmids except some forms of repeats.  Direct repeats can
be tolerated by coli but large inverted repeats not.  I am wondering 
where you got your DNA from as it is curious this was not noticed
in the other vector. If you PCRd this thing up then this could be your 
answer.  Some strange PCR product???  Just a thought!

________________________________________
Dan Gietz
Department of Human Genetics
University of Manitoba
770 Bannatyne Ave, Rm 250
Winnipeg, Man, Canada
R3E 0W3
GIETZ at BLDGHSC.LAN1.UMANITOBA.CA
_________________________________________




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