oswaldo at iats.csic.es
Tue Aug 29 07:03:11 EST 2000
Andrei Popescu wrote:
> premixed or mixed by me. Either I am doing something systematically
> wrong when mixing , or there is something more subtle. At 200 $, I
> cannot buy dNTPs every day. Has anyone found something similar?
Sounds like you are doing the typical "nucleotide screw-up" calculation.
If you mix the 100mM stocks from Promega in equal parts, what you get is
not 100mM each but 25 mM each in the nucleotide stock. From that, if
what you use is 0.2mM each dNTP in the reactions, this nucleotide stock
solution is 125x for the final reaction mixture.
But anyway, if this is indeed the case you have a suboptimal reaction as
a 4-fold decrease in nucleotides means no amplification at all. It
should not be so dramatic.
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