RT-PCR

Vance Baird 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

Dear Colleagues,
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





More information about the Arab-gen mailing list