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Primer concentrations with rapid PCR

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Tue Jan 4 05:12:53 EST 2000


In article <d515db7a49.root at freeuk.net>, Robert Hartley
<r.hartley at freeuk.com> writes
>In message <3870fdbe.5193211 at news.gwdg.de>

>          nvahsen at hot[nospam]mail.com    (remove the [nospam]) (Nicolas von 
>Ahsen) wrote:

>

>> Hi Lars,

>> 

>> I think you are right.

>> For a 20mer primer at 0.2 M Na-equivalents you can calculate a t1/2 of

>> approx. 3 sec. for annealing. And annealing time is should be short on

>> the LightCycler. More than 10 sec. makes no sense.

>
>

>
>10 sec annealing? 1sec if using capiliaries. 20 with tubes.

>
> Also I use a standard mix in my RapidCycler.9no optics)

>
>I beleive the increase is in Mg since the borosilicate adsorbs a lot of Mg.

>
>

>

Roderick Fuerst at Bio/Gene Ltd. is the guy to ask about RapidCycler and
LightCycler kinetics with regards primer concns. I'm sure he'll pop up
with the answer in the next day or so. Certainly annealing times are
very, very short on rapid cycling machines. On some 1sec is fast!

The Mg increase is due to the borosilicate. The large surface to area
ration of the capillaries takes out a lot of the Mg i.e. say 1.5mM out
of the 3mM that is the average Mg used by most users. 

Duncan
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk




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