help for my 5' RACE PCR!! Please!

Qunfeng Dong qfdong at iastate.edu
Tue May 27 22:34:56 EST 1997


Hi, any PCR experts,

  I've been trying to use 5' RACE RT-PCR to amplify a ~4.0 kb cDNA for
months. What I did was,

1. RT the first strand cDNA. I am pretty sure that this step worked well
because I used two internal primers (within the range of my ~4.0 kb target)
and they could amplify a correct fragment.

2. Get rid of excess primers either by microcentrifuge filtering or by using
Glassmax (from GibcoBRL), I think this step worked too because again those
two internal primers worked.

3. 2mM dATP + 1st strand cDNA about 10 minutes by TDT.

4. Then use oligo dT and the RT primer to do the PCR, I constantly got some
very small fragments (< 300 bps).

I use regular Taq for PCR, My guess is that 4.0 kb is just too large for
Taq PCR. Anybody has any idea that might help me? Any special PCR programs?
Thanks a million!!

Qunfeng
p.s Please send me a personal email, I am not a regular netter.
-- 
Qunfeng Dong
qfdong at iastate.edu



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