Make additional cycles with an already finished pcr product - (Aug/15/2011 )
hi, after the agarose-gel-electrophorese of my pcr products i realized that I have a very little yields. since more cycles increase the yiel I'm wondering if its possible to add more cycles to my already finished pcr. if yes, do i have to add again polymerase? Or can I just add 2 cycles more to my pcr?
Yes, you can take an aliquot of PCR product to run a gel and check the amplification, if needed, you can recycle the remaining reaction a few times without adding more Taq polymerase. If your PCR has already cycled for over 40 times, and need to cycle many more cycles, probably you need to add Taq.
My experience in re-amplifying PCR products is very poor, unless you use different primers than the initial reaction. Somehow, the reaction amplifies the short products, and the long (usually desired) product remains a minor component. It doesn't matter if you gel purify, at least for me.
I recycled my pcr reaction (two additional cycles) without adding more Taq polymerase. The bands on the agarose gel pic are now stronger and okay.
Thanks for your help!