Vance_Baird at QUICKMAIL.CLEMSON.EDU
Sat Apr 22 17:02:44 EST 1995
Subject: Time:5:27 PM
OFFICE MEMO RT-PCR Date:4/22/95
I'm going to do some RT-PCR using "short" (11- to 17-mers), degenerate (24 to
72 fold) oligonucleotides as primer pairs, or as an upstream primer in
conjunction with oligo-d(T)16.
Any suggestions on pCR settings? I was going to use the "low" Tm (A or T in
all variable positions) calculated for each oligo as the anneal-extend
temperature (range of 32 to 42 C). Is it necessary (would it be better) to
anneal and do a short-time (e.g., 0.5 to 1 min.) extension at the Tm then bump
up the temp. to the "standard" 60 C to complete the extension (for 1 min.)?
Total RNA (DNA free) will be the substrate. Must the total RNA be run over
CsCl to "clean" it? Is poly (A+)RNA a markedly better template/substrate? Is
it an absolute requirement?
As in the past, I greatly appreciate any guidance provided.
Vance_Baird at Quickmail.Clemson.edu
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