Cloning problems

Gregory A. Denomme 37_527 at uwovax.uwo.ca
Thu Oct 31 23:18:35 EST 1991


Greetins netters:
  I would like some help/advice with problems encoountered using PCR cloning.
I've been following the PCR threads - hopefully none of this is re-hashing.

We reverse transcribe total RNA to obtain specific cDNA for immunoglobulin V
region genes.  We then PCR amplify with a 5' leader primer and the same 3'
primer used in RT'n or one upstream.  This is followed by phenol extraction,
double restriction digests (Sac I and Xba I for example), then DNA gel
electrophoresis and band isolation by glassmilk.  Cloning is into pGEM11 (also
double digested) and transformation into DH5(alpha), plated on IPTG/X-gal.
Problems:
1.   Often the number of recombinants is *very* low.  Expect 100-200 cfu in
plating and get maybe 2-20 cfu's.
2.   White colonies almost always DO NOT have inserts of the expected size (we
are checking light blue colonies, blue centered colonies, etc).
3.   Some colonies do have the correct size but the sequence of these plasmids
is NOT a V region immunoglobulin gene.  A southern blot of the PCR amplified
product with a V region probe shows great hybridization.

Your thoughts would be appreciated and I will post a summary if need be.
Thanks,
Greg
Microbiology and Immunology, University of Western Ontario, London, CANADA
37_527 at uwovax.uwo.ca
gdenomme at uwovax.uwo.ca



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