HELP! My PCR stop working!!!!

Bruno Becker bbecker at iastate.edu
Fri Apr 28 22:36:00 EST 1995


In article <Pine.OSF.3.91.950428161206.25958B-100000 at leonis.nus.sg>,
Chee Yen Chin  <zoocyc at leonis.nus.sg> wrote:
>
>(Hi! Finally figure out how to post. :))
>
>Dear friends,
>
>I really need help this time. My PCR stop working, and I am desperate. I 
>tried all trouble shooting possibilities already, and I am at a lost now.
>
>A couple of weeks ago, we found a new pair of primers to replace our old 
>primers, and we know that the primers work since we got the primers from a 
>published paper. 3 weeks ago, our new primers arrived and it works well 
>for us under our conditions. About 1.5 weeks ago, our PCR stopped 
>working. Even positive control stop to show amplify band.
>
>I have tried the following:
>	1) changing to a new tube of dNTPs, 
>	2) using different brands of Taq polymerase
>	3) varying the MgCl2 concentration
>	4) using different Thermal Cyclers (have tried 3 brands already)
>	5) using my own homemade PCR buffer 
>	6) adding DMSO to my PCR mix since one of my primer is 57% GC content.
>	7) changing PCR conditions, including using the published conditions 
>	8) varying DNA concentration and using new tube and newly prepared DNA.
>	9) doing more cycles, hot start, warming thermal cyclers, etc, etc. 
>
>Basically, I tried every things that is within my understanding of PCR 
>with not much luck
>
>Please, can anyone give me some more suggestion?  I have run out of ideas.
>
>Your help will be greatly appreciated.
>
>Thanks
>
>Yen Chin CHEE
>Department of Zoology
>National University of Singapore
>( zoocyc at nus.sg )

Are you using a mix of your dNTPs, or are you adding your nucleotides
seperately from frozen stocks? I had some mysterious non working PCR
reactions until recently, when I stopped to premix the nucleotides.
Similar things have been reported here on the net by others (I dont
remember anymore by whom). My (maybe simplistic) explanation for this is
that perhaps the nucleotides bind to each other (as they do in double
stranded DNA): dCTP would thus be complexed by dGTP, dATP by dTTP. This
may not be a problem for the duration and at the temperatures of you PCR
reactions, but perhaps when storing that mix at freezer temp. for longer
times.

Of course, I have no proof for this hypothesis, therefore, take it with
a grain of salt!

Therefore, my advice would be: Do not prepare dNTP stock mixes for your
PCR! (but perhaps you dont, anyway).

Good luck!

-- 
Bruno Becker, Ph.D.
Email: bbecker at iastate.edu
Phone (USA): (515)294-3658
Fax (USA): (515)294-4141



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