More PCR questions

Rover Rover at livenet.net
Thu Oct 3 14:49:17 EST 1996


Your little ray of sunshine <eef1000 at cus.cam.ac.uk> 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

When we do this we generally add 10-30 uCi of a ~3000 Ci/mmol dCTP,
without changing the other parameters or nucleotide mixes. The
resultant prob is generally hot enough for any application.

The molarity of the dCTP is low enough as to not greatly influence the
PCR reaction. You could  make dCTP the limitting nucleotide in order
to get hotter PCR fragments but we have found that this really isn't
necessary.


SWB at Di1.evms.edu




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