bhjelle at vaxine.unm.edu
Wed Nov 6 13:36:03 EST 1991
In article <1991Oct31.231836.141 at uwovax.uwo.ca> 37_527 at uwovax.uwo.ca (Gregory A. Denomme) writes:
> 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.
>1. Often the number of recombinants is *very* low. Expect 100-200 cfu in
>plating and get maybe 2-20 cfu's.
This may be just that your cells aren't competent enough. I have, at times,
rescued an experimental bust by buying (mucho $$, yuck) BRL's >10e8/ug
HB101 or DH5. May be worth it sometimes.
My competent cells usually are around 10e6-10e7.
>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.
I presume your *clones* don't hybridize.
I tried an interesting variant on this last month. Made rapid minipreps
and spotted them onto nylon membranes, probed with an internal oligo
(biotin-labeled, detect with SA-HRP and ECL chemoluminescence reagents)
and picked clones on that basis. It is easier than restricting each,
and gives more information.
I suspect the procedure could be made even easier than that, if one
can bypass the miniprep step.
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