Stratagene cDNA cloning

NWHETT at ncsumvs.bitnet NWHETT at ncsumvs.bitnet
Fri Jul 26 14:52:00 EST 1991


Regarding Andy Mendelsohn's problems with cloning hemi-methylated
cDNA synthesized using Stratagene's method: I have made a couple
of libraries using this technique and not had much trouble with
high background (ie empty vector plaques). I got the kit before
they switched to the SURE strain, though; the version I have came
with the mcrA,B mutant PLKF' rather than SURE. I used gravity
flow Sepharose CL-4B columns to separate the cDNAs from the
adaptors, and had only 2% to 5% empty vectors, as determined by
picking random plaques, rescuing plasmid, isolating DNA and
checking for inserts.
Ross Whetten



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