PCR product stays on agarose gel well !! - I need help, what is it happening? (Jun/30/2009 )

I suspect your problem is two-fold: too much genomic template, and perhaps your primers don't work. You seem to be tweaking everything except the primer set -- template concentration, MgCl2 concentration, temperature gradients, cycle number, tubes vs. plate, etc. -- but how do you know your primer are any good?

I strongly suspect that if you temperature-cycle your 250 ng genomic DNA control (add everything but Taq, and make up this volume with water), you'll see the same blob of randomly annealed DNA.

You should go back to basics -- run a normal PCR with much less template, and a companion PCR using this same reduced amount of template and another primer pair, one close in Tm characteristics and amplicon size as your first reaction so you can run them on the same PCR program.

If this second reaction works, and the first one doesn't, I would conclude that one or both of your first reaction primers is faulty.

Conclusion to this story: The "big blob" of DNA inside the gel wells disappeared on 7/6/9. We still do not know what it was. That day I started a new tube of dNTPs, so my hypothesis is there was some contaminant in the reaction (presumably from the dNTPs).



We are still trying to get decent primers for our targeted amplicon, but that is another story. The only thing showing consistently is the positive control.