More PCR questions

Andrei Popov andrei.popov at bbsrc.ac.uk
Fri Oct 4 12:14:28 EST 1996


Your little ray of sunshine wrote:
> 
> I am trying to use 33P-dCTP in my PCR reactions and am having problems
> with the concentrations of dNTP's to use.  Does anyone have a working
> knowledge of this, and if so could they help me out?
> 
> Please email me direct .  Thanks a billion.
> 
> Em
> :-)
> Wellcome/CRC Institute

Dear Little Ray of Sunshine,

it depends on the application. If you want to label a probe:
I use 10-20 ng of template (PCR fragment) in the "normal" PCR buffer
plus 20 mkM (10% of normal conc) of dTTP, dATP, dGTP
and 5-10 mkl 32P dCTP (0.05-0.1 mCi; 3000 Ci/mmol). I use only one primer
to make one-stranded probe; thus, stricty speaking, this is not a PCR, rather
a cycling labelling reaction. Then I do 20-30 cycles with  very long extention
times (5 min at 72C)  to compensate for low dNTP concentration. Under these
conditions you should get 50-80% of 32P activity incorporated into
the probe.

Best wishes 

Andrei



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