Insert instability

[Martin Kennedy]

In article <9303242019.AA15489 at audrey.lmf.org>, jim at AUDREY.LMF.ORG (Jim Gale) writes:
> 
> 
> 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????

Jim,

Is your insert really unstable, or do you have a mixed prep of true recombinant
and vector DNA that just gives the impression you have unstable inserts?  In
other words, do you always see a mixed population of recombinant and deletion
derivatives in every colony after you re-transform another host, or do you 
just get lots of vector-sized plasmids?  If the latter is true, I'd suspect 
that your initial cDNA clone is contaminated with vector molecules.  You could
clean this up by streaking for single colonies from the glycerol stock and 
then using severl of these for minipreps. 

> 
> 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
> 

I have a flyer from a company called "Kontes" that supplies just these items,
and a little motor to drive the pestle if necessary.  Sorry, but they don't
give an address, although the flyer is US published, and our agent for them is
a local company not represented in the States.


Cheers,

Martin

Martin A Kennedy (E-mail = cytogen at chmeds.ac.nz)    
Cytogenetic and Molecular Oncology Unit               
Christchurch School of Medicine                       
Christchurch, New Zealand