RT-PCR difficulty - PLEASE HELP!!!!!
jpcd0 at mole.bio.cam.ac.uk
Thu Aug 22 07:36:35 EST 1996
In article <Pine.OSF.3.93.960821121627.6082B-100000 at alpha.Colorado.EDU>,
Tonja Burshek <burshek at ibg.colorado.edu> wrote:
> Background: We are trying to reverse transcribe (following DNase
> digestion and clean-up) using an anchored poly-T primer, followed by PCR
> using the same poly-T paired with a 10-mer.
> Upon many moons of optimization IT WORKED BEAUTIFULLY!!!
> Problem: after many positive repeat trials (with various 10-mers and
> several different RNA and RT preparations), it QUIT working!!! None of
> the new combinations OR any of the combos that worked previously show
> ANYTHING on the gel!! Occasionally, the LAST sample of 20 will work, but
> if 20 of the same sample are tried, NONE come out (even if the one sample
> was the one that came out before).
> We are STUMPED. Any suggestions will be greatly appreciated!!
> Thank you very much in advance,
> Tonja & Elizabeth
Why are you using such tiny primers? Your annealing temp for PCR must be
very low - and dont you risk non-specificity with such a small primer?
What conditions did you finally settle on for this procedure?
Lets have a little more info!
We do RT PCR with dT(15-18) followed by two PCR primers (24-30bps) and
then do nested PCR with two Gene specific primers with the two primers
outside the dT stretch. Doesn't always bring up clean bands but probing
with an internal GS primer usually identifies the band of interest.
John Dixon Lab 44 (1223) 334131
Wellcome/CRC Institute Fax 44 (1223) 334134
Department of Genetics
United Kingdom e-m: jpcd0 at mole.bio.cam.ac.uk
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