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dNTP concentration in PCR

Robert Horton horton at molbio.cbs.umn.edu
Thu Jul 28 21:30:52 EST 1994


Patty McManus (mcmanusp at student.msu.edu) wrote:
: For folks who are having PCR problems, are you sure your dNTP
: concentration is correct?
: 
: Maybe no one out there is as big a bozo as I, but my quality of PCR 
: ( and quality of life) improved dramatically when I realized my dNTP
: concentration was four times to high!
: 
: I won't go into the details, but it has to do with the fact that when
: you combine 20mM stocks of each of 4 dNTPs, the final concentration of each
: is 5mM and not 20mM.  Pretty obvious, I know, but sometimes the obvious
: is overlooked.  My apologies to anyone whose intelligence was insulted.

So, was the dNTP too high or too low?

If it's too low, presumably the polymerase "starves", and if it's too
high, the dNTPs chelate too much Mg++, so there's not enough left for
the enzyme (or so I'm told). But, if you take the [Mg++] into account,
I imagine you could successfully vary the [dNTPs] quite a bit. 

Anybody care to post some data regarding the limits of allowable
[dNTPs] in PCR?  My neighbor does differential display (in which, for
the first round they only need to detect the product on an autorad) at
2uM each.  I guess you don't get EtBr-stainable bands with this
concentration, but he says you do with 20uM. So maybe a lot of us are
using an order of magnitude more dNTPs than we need to. Waste not, want
not...  (it might also affect error rates)


Bob Horton (Ph.D.!)   /\ "Crash programs fail because of the theory that
U. of Minnesota, CBS  || with nine women pregnant you get a baby a month" 
1479 Gortner Ave.    /||\   -Werner von Braun.  Disclaimer:"Bob who?"
St. Paul, MN 55108    ^^   horton at molbio.cbs.umn.edu/(612) 624-3790



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