bluescript


Tue May 17 16:33:05 EST 1994


Dear Neil, I showed your letter to my postdoc. We had a problem with a
ZAP library and that was that when she tried to cut out the inserts, there
were none. We also had had a very good set of pure plaques and were
pretty irritated. In addition, she had done alot of screening-in humans
and also yeast and so I knew it wasn't her technique. In fact you might
have seen an email message of mine. Someone suggested that we try different
enzymes (cloned in RI) and sure enough if we cut outside of it we got
the insert and they did have the gene. I know your problem is different, but
back in the "old days" of the early 80's when I was in grad school, I had
a blot that had an insert prep on it. This was when nick translations only
were done and I used a whole plasmid. I found out I couldn't do this because
there was enough residual of the vector sequence on the blot, even thoughit
was "purified insert" that only it hyb'd. The insert sequence didn't! Even
when I nick translated the insert itsself, the vector still hyb'd. When I
mentioned that to my postdoc, she suggested that you just have a small
unexcised population mixed in there and that is what is hybing. I have no
idea, just a thought. But don't throw those guys out until you're sure
they are not what you want. So, be careful about your probe and hyb 
conditions. I suspect that someone from Stratagene will see this on the
network eventually (they saw my message) and jump to offer suggestions.
Sandy Berry-Lowe in Colorado



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