RT-PCR or purchase cDNA library?

Steven Munger stevem at aug.com
Tue Oct 15 14:45:52 EST 1996


In article <goldberg-1410961545410001 at 140.176.74.203>, 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
> factor)?
> 
> Thanks for any responses.
> 
> Steve


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.

another Steve



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