help for my 5' RACE PCR!! Please!
scole at DARWIN.SFBR.ORG
Wed May 28 12:35:57 EST 1997
Do you need a full-length cDNA clone? Otherwise, could you use sequence
information that you might already have, or could get from sequencing the
cDNA fragment that you have amplified with internal primers, to design a
reverse primer much closer to the 5' end?
You might also try using your internal reverse primer as a nested reverse
primer for your PCR, instead of the RT primer. Your RT primer might have
primed some non-specific cDNAs in the RT reaction, which would be shorter
and preferentially amplify in the PCR reaction.
Shelley Cole, Ph.D.
SW Foundation for Biomedical Research
San Antonio, TX
On 28 May 1997, Qunfeng Dong wrote:
> 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!!
> p.s Please send me a personal email, I am not a regular netter.
> Qunfeng Dong
> qfdong at iastate.edu
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