arms PCR problem - (Jul/14/2008 )

Hi all,

Im new to this forum.... but not new in the field. But Im having some problems with an ARM-PCR reaction Im trying to make it.... I have the primer sequences and PCR program from an article... check in BLAST the primers... are OK.... but with the program I have some problems....

its like this:
96C-1min
95C-25sec, 61C-45sec, 72C-45sec - 5 times;
96C-25sec, 65C-50sec, 61C-45sec - 21 times
96C-25sec, 55C-60sec, 72C-120SEC - 4 times;

I have tryed this program, changing the Mg, primers and DNA concentration, but nothing. Tryed more with a "clasic" PCR program, varying the different conc. but nothig....

Can anyone help me with an advice PLSSSS!!!

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Hi Georgina,
can please provide more details: Taq enzyme used, amplicon length, template origin, rxn conditions (Mg2+ conc., primer conc., ...) ...
Maybe you used a hot-start Taq (e.g. AmpliTaq Gold, FastStart or Platinum) which needs a activation step (e.g. 5-10min @95°C) or wrong Mg2+ conc..
Maybe with more information your answer is easier to answer.
Let us know

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I have used GoldTaq from Promega.... amplicons should be 78pb, template is human DNA from EDTA blood and I want to amplify a region containing a Fok I polym.; the conditions they have described were with 6mM allele primer and 3mM consense primer.... which it seems very highy to me... but hey say they had results... and Mg... I have tryied every conc from 1-8mM final conc and nothing. My only idea that I still have is to try other primer conc.... ie: 2mM allele primer/1mM consence primer and 1/1mM conc.... after that.... dont know

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