perkin elmer vs light cycler
Nicolas von Ahsen
nvahsen at hotmail.com
Thu Sep 2 13:15:50 EST 1999
On Wed, 1 Sep 1999 22:21:48 +0200, "Gys de Jongh"
<GysdeJongh at compuserve.com> wrote:
>Dr. Duncan Clark <Duncan at nospam.demon.co.uk> wrote in message
>news:yfkUmFA3P$y3Ewie at genesys.demon.co.uk...
>> In article <7qgptv$8ee at news.service.uci.edu>, Joan Marie Shields
>> <jshields at rigel.oac.uci.edu> writes
>> >Is the MgCl2 there for the polymerase
>> The polymerase, but an equimolar amount is titrated out by the dNTPs
>> i.e. 0.2mM each dNTP takes out 0.8mM MgCl2 leaving 0.7mM for the enzyme.
>i agree with you duncan. Two points to mention i think 1) if the template is
>in TE than further Mg is lost by EDTA in the TE ( 2 Mg for 1 EDTA) and 2)
>the pure physical effect. Mg is a divalent cation. The shielding effect of
>divalent cations is a factor 10 higher than that of monovalent cations like
>Na. So 1mM Mg has the same effect as 10 mM Na. In this respect the effect of
>Mg is the same as lowering the annnealing temp , adding DMSO etc. So if the
>Mg is raised above the optimum for the polymerase enzyme (see duncans
>advice) then you may get loss of specificity or better results in the case
>of GC-rich templates.
you are right, but Mg has a 140x(!) times higher stabilizing effect on
(see: Nakano S, M. Fujimoto, H. Hara, and N. Sugimoto. Nucleic acid
duplex stability: influence of base composition on cation effects.
Nucleic Acids Res. 27 (14):2957-2965, 1999).
And regarding the original post I do very much think that rapid
cycling has many advantages and that it should be possible to adopt
virtually ervery PCR to it.
But anyone explain me this: I have a PCR which amplifies only w/o SYBR
green in it. Same mixture, same template and same PCR run using a
capillary with SYBR-green and I get no amplification. I will
investigate a little further in this and if nothing helps I'll do a
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