Michael L. Sullivan
mlsulliv at facstaff.wisc.edu
Tue Aug 29 09:51:19 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.
I think this is correct. Some protocols in fact call for using on the
order of 50 uM dNTP, supposedly to reduce misincorporation. I recently
compared 200 uM and 50 uM dNTP with Amplitaq LA, and although there was
clearly a reduction in product at 50 uM, I still got plenty of product.
Something I would check would be the pH of the dNTP stock using pH paper.
pH should be >7, I think. If the solution is acid, I think the dNTPs
disproparionate into dNDP and dNtetraP.
Michael L. Sullivan, Ph.D
U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706
(608) 264-5144 Phone
(608) 264)-5147 Fax
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