32P-dNTP in PCR

Minsoo Yoon m.yoon at auckland.ac.nz
Mon Dec 20 22:41:56 EST 1999


in article 385870CD.C79 at usm.edu, Shiao Wang at Shiao.Wang at usm.edu wrote on
16/12/99 4:55 PM:

> I would like to incorporate some 32P-labeled nucleotide in a PCR
> reaction. The purpose is to show that the PCR is very specific by
> over-exposing the dried gel to film. I'm using Redivue 32P-dATP from
> Amersham. Using the same mastermix of PCR components, one without
> 32P-dATP and the other with 0.5 ul of 32P-dATP in a 50ul reaction, the
> one with labeled nucleotide produces significantly less amplified
> product in a EtBr stained gel at annealing temp above 60C.
> 
> My hypothesis is that there is something in the Redivue stabilizer that
> breaks down at elevated temperature and inhibits DNA pol. Does anyone
> have similar experience? I called Tech Support at Amersham and the
> support person said that Redivue 32P-dATP works well with Klenow at 37C
> but that they have no data at higher temp because they never used it in
> PCR.
> 
> More important question: can someone with experience in labeling PCR
> products, especially RT-PCR, please let me know which 32P-dNTP (with or
> without stabilizer and specific activity) and which DNA pol worked?
> We're using Tth in one-step RT-PCR and perhaps the Tth is the problem.
> 
> Thank you for your attention.

Reduce the amount of cold dATP down to at least 1/10. The ratio between the
cold and hot dATP would still be considerably high but that will give you
strong bands.   From my experience, I don't have any preference for any
particular hot dNTPs or polymerase.  Warning; the radioactivity of each band
won't be quantitative nor proportional to the amount of template.  I suggest
you to define the linear dynamic range first, which is the most critical
step of all, I guess.

Good luck
Minsoo 





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