RT PCR of long template

Pow Joshi pow.joshi at gmail.com
Tue Oct 3 14:46:31 EST 2006


On 9/29/06, DK <dk at no.email.thankstospam.net> wrote:
> In article <mailman.1240.1159570040.20007.methods at net.bio.net>, "Pow Joshi"
> <pow.joshi at gmail.com> wrote:
> >Hi,
> >
> >I am trying to clone a 3.2 kb cDNA with RT PCR .... while reading on
> >the topic, I realised that the PCR might get difficult due to the
> >sheer length (assuming I have very high quality RNA :)....
> >I would appreciate any input on strategies used for this type of cloning.
>
> Worked for me once (N=1):
>
> Do RT-PCR under variety of conditions --> run high quality gel and
> cut out pieces corresponding to the expected product size, even if you
> can't see anything on a gel --> extract DNA from that "band" and use
> it as a template in PCR reaction (do it with 3 different template amounts,
> each +/- 5% DMSO in the reaction) -> if all goes OK, there will be
> at least one reaction in that whole grid that has the product you desire
> clearly observable on a gel.


Thank you, Dima,  and everyone else for the input. Will keep you
posted on how the cloning goes.

pow
>
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