RE- RT-PCR

Vance Baird 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

Dear Colleagues;
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.

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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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