Vance_Baird at QUICKMAIL.CLEMSON.EDU
Tue May 2 10:03:36 EST 1995
Subject: Time:10:30 AM
OFFICE MEMO RE: RT-PCR Date:5/2/95
The following is a compilation of the responses I received from my request for
help in setting up RT-PCR experiments using short, degenerate oligonucleotides
as primers. When I get it to work, I'll add my own helpful hints!
just a simple suggestion on the PCR.
I never did a RT-PCR, but once you have a synthesizd the cDNA,
it should be like a "normal" PCR.
One suggestion is to try the Touch-down PCR (decreasing
annealing temperature in the first few cycles). For a reference see
Don e al., (1991) N.A.R. 19:4008.
The other is to use a hot start, that is adding the polymerase
after a initial long denaturation (say 4' at 95) while holding
the samples at 85 C until all of them received the polymerase.
For a reference about this and other useful info, see:
Hosta and Flick (1992) USB newsletter "comments" vol 18, n. 3, p1-4.
I'm doing some RT-PCRs without alot of success and so any advice would be
welcome for me as well.
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