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

Zhonglin Chai Zhonglin.Chai at baker.edu.au
Sun Oct 28 18:55:37 EST 2001


For the purpose of your experiment ("sequence it to see if it's the right thing"), you may just clean up or gel purify your pcr products and sequence it directly using either or both the primers used for your pcr amplification. We have purified using Qiagen's Gel Extraction Kit and sequenced a pcr fragment as short as ~150bp without problem. 

Zhonglin Chai, PhD

Molecular Hypertension Laboratory
Baker Medical Research Institute
P.O. Box 6492
Melbourne 8008
Victoria, Australia
Telephone: +61 3, 9522 4357
mobile : 0413 58 1940, or international +61, 413 58 1940
Fax: +61 3, 9521 1362 
email: zhonglin.chai at baker.edu.au


>>> Mike Levin <mlevin77 at mediaone.net> 10/29/01 09:41am >>>
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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