3 replies to this topic

[Shrubal]

Hello!

I was wondering if anyone could explain the specific role of water in a PCR reaction. I was wondering if the addition of water could be completely eliminated and instead compensated by adding more template if I had a very low concentration of template.

Thanks!

[Trof]

Depends whether you have your template disolved in water or in yogurt.

[hobglobin]

Water is the solvent you use to get the final concentrations of the reagents in the desired volume...if you use more diluted DNA or other reagents (Mg²⁺, primers, buffer) you might end up that you don't need to add any additional water, but that's by chance then, as you cannot change their volume (except DNA often-times).
Anyway there's no need to add additional water as the water can come from any other source, but surely it's much easier to use it to get the desired volume.

[mightymus]

Even I was having the same problem of low tempet.Try to evoporate the solvent (water or any buffer your dna is suspended) by heating the tube at around 55-60 C  and increase the concentration of your dna. I did it a couple of times and it worked perfectly for my qpcr (:. By this way you retain the same final volume as you used to do before.

PS: make sure that the cap is open when heating