dNTP problem

[Michelle Gleeson]

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.

cheers,
Michelle

CGGATCGAAACTATT    Michelle Gleeson    CAGGGCTGAATA
   Research Scientist, Molecular Parasitology Unit
     University of Technology, Sydney AUSTRALIA
GCCTAGCTTTGATAA                        GTCCCGACTTAT

Andrei wrote:
>Hello everybody,


>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 
>similar?


>Thanks in advance!


>Andrei Popescu, RA Molecular Biology Vanderbilt University




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