Insert instability

braam at DINGO.RICE.EDU braam at DINGO.RICE.EDU
Wed Mar 24 13:22:38 EST 1993


>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


We have found that DH5-alpha (recA-) work for cloning our unstable insert, 
while Stratagene Sure cells recombine the plasmid consistently.

hope this helps.  
janet





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