PCR from genomic dna
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Wed Jun 14 03:50:10 EST 2000
In article <rhubner-2F0E06.21401413062000 at news.uia.ac.be>, Roland Hübner
<rhubner at gins.uia.ac.be> writes
> Well, when the concentration of dNTPs or primers is altered, the
>concentration of free Mg++ is affected...
I don't have a problem with dNTPs affecting the Mg++ and obviously it is
critical to get that right. Taq likes about 0.7mM of free Mg++ so with
200uM dNTps one needs a minimum of 1.5mM Mg in a PCR reaction. With
primers I've never noticed any effect. However that may be because we
don't go sticking in vast amounts of primers so it has never been an
issue. Normally we are using 0.1-0.5uM depending upon what we are doing.
I was more interested in why additional template above the norm. Would
take out Mg and how much it might take out.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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