michelle_gleeson at hotmail.com
Tue Aug 29 17:16:53 EST 2000
Hi Andrei and everyone,
It's been quite a while since I used this type of dNTP, but looking back at
my notes, we prepared them as follows:
1 in 10 dilutions of 100 mM stocks, in milliQ water = 10 mM each.
Equal amount of each 10 mM dNTP mixed to create working stock
4 ul working stock added per 50ul PCR rxn
I haven't re-done the maths, but if you believe my honours thesis this is
0.2 mM of each dNTP final concentration.
I recall we made relatively small amounts of working stock at a time, to
avoid too many freeze-thaw cycles. I also recall we always ran out of one
100 mM stock before the others. Because of this (and to reduce costs) we
switched to a polymerase supplier which provides tubes of pre-mixed, ready
to use dNTP for free with the Taq. If they do it down here, I'm sure the
same kind of deal is available in the US. Never had a problem with the
'free' dNTPs, they even worked for long PCR.
And it's much faster to bung on a PCR at the last minute.
CGGATCGAAACTATT Michelle Gleeson CAGGGCTGAATA
Research Scientist, Molecular Parasitology Unit
University of Technology, Sydney AUSTRALIA
>I have been trying to get a PCR started for 1 month, no results. I modified
>everything, especially annealing temperatures and DNA concentration. I took
>the template and primers to another lab, and >boom, the reaction worked in
>4 out of 4 samples. I came back and repeated in my lab but using dNTP
>mixture from another lab, and it worked beautifully. I tried in parallel
>with my old dNTP-nothing, not even the positive controls. I bought new dNTP
>from promega, 4 vials - nothing again. I used same .25 mM concentration
>each dNTP, in successfull or unsuccessfull reactions. So it seems to depend
>on whether the dNTPs are 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
>Thanks in advance!
>Andrei Popescu, RA Molecular Biology Vanderbilt University
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