3 replies to this topic

[polymerase1986]

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?
Thanks

[pcrman]

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.

[phage434]

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.

[polymerase1986]

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!