RT-PCR difficulty - PLEASE HELP!!!!!
burshek at ibg.colorado.edu
Wed Aug 21 13:22:32 EST 1996
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
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