RT-PCR question

Shahram Mori smori at opus.nmsu.edu
Thu Oct 6 01:19:00 EST 1994


Annette C. Hollmann (ah690549 at mbcr.bcm.tmc.edu) wrote:
: In article <Cx24Ep.4r5 at ns1.nodak.edu> santoso at badlands.NoDak.edu (Adi Santoso) writes:
: >i try several time RT-PCR with 3' primer using oligo dT (16 mers) just 
: >never work. I check all the reagents and template RNA, they are all 
: >oke...!!! any suggestion for me.  I just almost give up with it..
: >
: >ADI

: I had the same problem, using the Perkin-Elmer GeneAmp kit.
: I ended up switching to random hexamers for the RT step.
: Perkin-Elmer techies have suggested that anchoring the dT tail with the
: last base of the gene might help.

: They also suggested that I make sure that I do the initial 10 minute
: incubation at 20 to 25 C, and that I increase the RT time from 15 min to
: 60 min.

: If you are using the Perkin-Elmer kit and have no particular reason to
: use the dT primer, I would suggest switching to random hexamers.

: Annette
: ah690549 at mbcr.bcm.tmc.edu

: Good Luck

I agree completely with Annette. Use 6mers for cDNA synthesis ( 1hr at
25C) and forget oligo dt's. Make your library using random 10mers. 
cheers,

--
Shahram Mori					   _/\_
Program in Molecular Biology			  _\  /_
Dept. of chemistry and Biochemistry Box 3C	  \_  _/
NMSU  Las Cruces NM				    ||
88003





More information about the Methods mailing list