RT-PCR difficulty - PLEASE HELP!!!!!

Taek H. You tyou at postbox.acs.ohio-state.edu
Sat Aug 24 01:00:09 EST 1996


In article <Pine.OSF.3.93.960821121627.6082B-100000 at alpha.Colorado.EDU> Tonja Burshek <burshek at ibg.colorado.edu> writes:
>Path:
>magnus.acs.ohio-state.edu!csn!nntp-xfer-1.csn.net!boulder!alpha.Colorado.EDU!bur
>shek
>From: Tonja Burshek <burshek at ibg.colorado.edu>
>Newsgroups: bionet.molbio.methds-reagnts
>Subject: RT-PCR difficulty - PLEASE HELP!!!!!
>Date: Wed, 21 Aug 1996 12:22:32 -0600
>Organization: University of Colorado at Boulder
>Lines: 18
>Message-ID: <Pine.OSF.3.93.960821121627.6082B-100000 at alpha.Colorado.EDU>
>NNTP-Posting-Host: alpha.colorado.edu
>Mime-Version: 1.0
>Content-Type: TEXT/PLAIN; charset=US-ASCII
>X-Sender: burshek at alpha.Colorado.EDU


>        Background:  We are trying to reverse transcribe (following DNase
>digestion and clean-up) using an anchored poly-T primer, followed by PCR

Is this poly-T a RNA primer or you mean a poly-dT primer? 					   

>using the same poly-T paired with a 10-mer.

Can you use the same primer in PCR as in RT?
It must be a poly-dA primer to be annealed to the poly-T sequence used in cDNA 
synthesis.

>        Upon many moons of optimization IT WORKED BEAUTIFULLY!!!

If I am correct, that the beautiful product might turn out to be the 
ugly nonspecific (unwanted) product.

Overall, am I missing anything or misunderstanding?





More information about the Methods mailing list