PCR reamplification - (Sep/07/2010 )
I have two general questions:
- Does anybody know why is sometimes so difficult to reamplify a PCR product with the same primers, and sometimes it is easy?
- Why is a nested PCR usually better in order to reamplify? What happens at the end of the PCR product that makes it easier for a nested primer to anneal?
Thanks in advance!
There are many reasons for this. One is that if you do not clean the first PCR reaction (i.e. remove old primers, etc), then it is likely that the old PCR components will inhibit the amplification of the PCR product when you try to re-amplify. This is simply a result of the second PCR reaction having a very different composition compared to the first. To get around this simply purify the PCR product before re-amplifying.
My two cents
Thanks for your quick reply, but I forgot to say that I usually clean my PCR product with a Qiagen kit (MinElute PCR purification kit), so I guess that in my case, this is not the problem.