RT-PCR or purchase cDNA library?
stevem at aug.com
Tue Oct 15 14:45:52 EST 1996
In article <goldberg-1410961545410001 at 18.104.22.168>, goldberg at bms.com
(Steven Goldberg) wrote:
> I am planning to screen a rabbit liver library for a gene. I have
> sufficient information to make RT-PCR feasible and can buy poly A RNA
> prepared from this organ. Or I could get the cloned cDNA library and use
> it. The poly A RNA is about 5-fold less expensive than a library, which
> is a consideration, but I have not had any experience with RT-PCR. If
> someone has used this technique I would be interested in hearing your
> opinion and a recommendation for a kit if you were pleased with the
> results. Is RT-PCR sufficiently reliable that someone who is familiar
> with PCR can use it without a long learning curve (time is somewhat of a
> Thanks for any responses.
If you are familiar with PCR, then RT-PCR is not too big a jump. it is
especially useful if you are dealing with a rare message. If you haven't
done PCR,then I would stick with the library (for time reasons). Library
screening (with plaque hybridization) does give you the advantage of
avoiding the polymerase-induced mutations often seen in PCR products, and
can allow you to pull out the full-length cDNA in one big chunk. I never
use kits for the RT-PCR (you can buy the necessary components separately
for less), and would be glad to give you my protocol, which works
consistently with a vairety of primers (exact and degenerate) and
templates. For reagents, I would recommend BRL's Superscript II MMLV
reverse transcriptase. Also, for the PCR, I use Taq polymerase spiked
with pfu polymerase (60:1, units:units)...this gives efficient
amplification with fewer errors.
One other consideration...if you are trying to amplify a product larger
than 7-8 kb, RT-PCR will be much more difficult. Feel free to email me,
and I would be glad ot send you my protocols.
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