Cloning problems

[Brian Hjelle]

In article <1991Oct31.231836.141 at uwovax.uwo.ca> 37_527 at uwovax.uwo.ca (Gregory A. Denomme) writes:
>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.

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.
>
Brian