RTPCR

Wolfgang Schechinger marsupilamiwolfgang at web.de
Fri Sep 20 10:29:22 EST 2002


Dear Bina, 

why do you want to re-invent the wheel? Better get say 10 independent papers you can access for free (from PubMed, of course :) and look which primers they are using. I think, GAPDH as control is so well established that you first should rely on published information. Even if your software says some primers are ok, you still need to test them (there still is a slight chance of failure) and you have to establish working PCR conditions in your lab (with your cycler and reagents - already enough work to do).

Roche or other companies who offer realtime equipment could be worth searching their websites for protocols, too.

Regards,

Wo

roybina1 at yahoo.co.in (bina roy) schrieb am 20.09.02 16:30:10:
> Dear All,
> 
> I am a beginner of this method of RTPCR. In the first instance I tried
> to look for a sequence of human GAPDH from pubmed and pasted it to the
> PRimer3 program to choose primers. My problem was
> 1)I was confused as to what information should be given to Primer 3,
> the mRNA CDS or the whole gene sequence (and the CDS from there)
> itself.
> 2)Primer 3 gave 5 outputs (because I chose 5). Then should the sense
> and antisense primers be taken from the region spanning the CDS.
> 3)Furthermore, I want to know that if I could feed the Primer 3 output
> into any other software and screen for the best ones (mostly to avoid
> primers that will encourage primer-dimer formation).
> 
> Thanks in advance for your help.
> 
> Bina.


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