Originally Posted by jiajia1987
I am sorry to add to this, but I do not get why the concentration of the PCR product in the reaction mixture would be a problem. If the primer used in the first PCR anneals to the template and makes a PCR product, the PCR product should be similar to the template, with the ends similar to the primers used in the first PCR, right? Since that is the case, why would the second PCR be a problem? I am confused.
Well the first PCR makes millions of copies (maybe tens of millions). These copies will 1) Bind up Mg++, so your optimized PCR is now messed up because of the ratio of Mg++ to DNA, 2) Primer now has more target sites to bind to, but the DNA polymerase has all that PCR product in the way of itself and the template 2a) given that DNA polymerase finds the PCR-template everytime---the primer concentration (eg 20nM) will probably be used up in the first few cycles of PCR amplification (probably 4-5 cycles)--now definitely your Mg++ conc will be overwhelmed. Same thing for the buffer capacity, and possible inhibition of Taq from just too much DNA in reaction.