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

Tonja Burshek burshek at ibg.colorado.edu
Wed Aug 28 09:05:04 EST 1996



On Wed, 28 Aug 1996, Taek H. You wrote:

> 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
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> 
> >In article <Pine.OSF.3.93.960821121627.6082B-100000 at alpha.Colorado.EDU> Tonja
> >Burshek <burshek at ibg.colorado.edu> writes:
> >>Path:
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> >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
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> 
> >>        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?
> 
NO!  the other primer is a non-specific random decamer.  We expect between
50 and hundreds of bands.  It is the last step of a differential display
experiment.  Thank you for any imput.






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