RT-PCR question

Tracy Aquilla aquilla at salus.med.uvm.edu
Tue Oct 4 18:44:57 EST 1994


In Article <36s199$647 at netnews.upenn.edu>, smoore at mail1.sas.upenn.edu (Sean
David Moore) wrote:
>Adi Santoso (santoso at badlands.NoDak.edu) wrote:
>: i try several time RT-PCR with 3' primer using oligo dT (16 mers) just 
>: never work. I check all the reagents and template RNA, they are all 
>: oke...!!! any suggestion for me.  I just almost give up with it..
>
...(deleted)...
>If all of the reagents are "ok", then the reaction will work..unless the 
>target isnt there.   
>

Not if the wrong PCR profile is used. If you tell us more about the
parameters of your experiment, maybe someone can help. I think a 16mer of
oligo dT has a pretty low Tm (about 32C at 50 mM Na conc?). I suggest using
a longer primer, and one that's specific for your gene, instead of the dT oligo.



Tracy Aquilla, Ph.D.
Molecular Physiology and Biophysics
University of Vermont
aquilla at salus.med.uvm.edu



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