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

Taek H. You tyou at postbox.acs.ohio-state.edu
Tue Aug 27 23:31:33 EST 1996


In article <tyou.320.321E9A69 at postbox.acs.ohio-state.edu> tyou at postbox.acs.ohio-state.edu (Taek H. You) writes:
>Path: magnus.acs.ohio-state.edu!ts30-16.homenet.ohio-state.edu!tyou
>From: tyou at postbox.acs.ohio-state.edu (Taek H. You)
>Newsgroups: bionet.molbio.methds-reagnts
>Subject: Re: RT-PCR difficulty - PLEASE HELP!!!!!
>Date: Sat, 24 Aug 1996 06:00:09 GMT
>Organization: The Ohio State University
>Lines: 36
>Message-ID: <tyou.320.321E9A69 at postbox.acs.ohio-state.edu>
>References: <Pine.OSF.3.93.960821121627.6082B-100000 at alpha.Colorado.EDU>
>NNTP-Posting-Host: ts30-16.homenet.ohio-state.edu
>X-Newsreader: Trumpet for Windows [Version 1.0 Rev B final beta #1]


>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!bu
>r
>>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?


I've made a mistake on this.
I only thought about PCR, not considering RT reaction.
Did you used a specific primer in PCR?





More information about the Methods mailing list