RT-PCR Help Me :)

Bill BILL at Pharm.SOM.sunysb.edu
Sat May 22 10:34:27 EST 1993


Hi Steven;

It is a good idea to DNase treat your samples prior to RT.  I
have found this to be espesially true with tissue samples.  We
generally use AMV-RT from promega and perform the RT at 42C on 1
ug of poly A+ RNA.  If you are using oligo dt as the RT primer
I generally use 50ng of a 12-18mer, however lower
concentrations may yield more full length cDNAs.  For most RNAs
that I have looked at I then dilute the RT mix 1:50 and place a
ul in the PCR rxn.  Your PCR conditions will depend upon the GC
content of your primers, but my guess is that the conditions you
used should work although you might want to try raising the
annealing temp.  The shorter the times the better especially
since you only have a 130 bp prpduct (30s at each should be
fine.
As a side note: we have found that when the same primer is used
in RT that is used in the PCR we sometimes can not get a product
(go figure).  This is not true for all primers but we have seen
it with quite a few.

Hope this helps

Bill Richards
Pharmacology
SUNY at Stony Brook













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