RT-PCR question

Annette C. Hollmann ah690549 at mbcr.bcm.tmc.edu
Tue Oct 4 17:01:06 EST 1994


In article <Cx24Ep.4r5 at ns1.nodak.edu> santoso at badlands.NoDak.edu (Adi Santoso) writes:
>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..
>
>ADI

I had the same problem, using the Perkin-Elmer GeneAmp kit.
I ended up switching to random hexamers for the RT step.
Perkin-Elmer techies have suggested that anchoring the dT tail with the
last base of the gene might help.

They also suggested that I make sure that I do the initial 10 minute
incubation at 20 to 25 C, and that I increase the RT time from 15 min to
60 min.

If you are using the Perkin-Elmer kit and have no particular reason to
use the dT primer, I would suggest switching to random hexamers.

Annette
ah690549 at mbcr.bcm.tmc.edu

Good Luck





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