Magnesium in PCR - (Jul/14/2009 )
I've been searching around (perhaps not in the right places) and can't seem to figure out the answer to my question. Is Magnesium consumed/used up in reactions? I have a protocol where the sample is taken through multiple reactions (poly A tailing, second strand synthesis, then PCR) with no purification in between, and was wondering if I need to worry about Magnesium accumulating, or if it's like dNTPs and is (mostly) used up in each reaction.
No, it is all still there. You should either purify between reactions, modify the buffers added to give the correct buffer concentrations, or dilute the product between reactions sufficiently that the amount of the previous buffers present at the next step is insignificant.