RT-PCR or purchase cDNA library?

Gail Otulakowski gotulak at sickkids.on.ca
Tue Oct 15 14:09:27 EST 1996


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.
> 
> SteveRT-PCR will be much quicker to yield your clone than library screening.  
Almost any of the commercial kits are okay.  The warning needed is that 
Taq polymerase is not very accurate - if you use Taq you should sequence 
2 or 3 clones to be sure no misincorporations have occurred.  There are 
some "proof-reading" thermostable polymerases around you could try that 
should give fewer errors (e.g. Pfu).  I have not tried them much - it 
seems to be more difficult to optimize the reaction conditions for them, 
from the little experience I have with them.

Good Luck



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