advice needed: my PCR fragment gives low yield when grown up! Why?

Mike Levin mlevin77 at mediaone.net
Sun Oct 28 17:41:28 EST 2001


Hi everyone -

   I'm looking for ideas on the following problem. A post-doc in my lab did
PCR to clone a fragment (by homology) of a flatworm gene. He got one - it's
the predicted size (about 200 bp), and he now wants to get it into a vector,
grow it up, and sequence it to see if it's the right thing. So, he ligates
it into pBluescript (a high copy number plasmid), transforms bacteria, and
does a miniprep on all the colonies (18 of them). All the bacteria grow fine
on Ampicillin-containing media. But here's the weird part: while the
vector-only control gives a nice plasmid DNA yield (suggesting his miniprep
procedure is fine), all of the insert-containing ones yield very little DNA.
There's just barely enough to see that there's even an insert in there. A
repeat of the procedure gave the same result. What could be going on? The
fragment is small, so I wouldn't have thought that it would cause the
plasmid not to replicate. Are there any common techniques people would try
at this point (maybe a different vector, etc.)? Any ideas on what could be
going on are greatly appreciated. Please email to mlevin at forsyth.org.
Thanks!!

Mike Levin





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