2 replies to this topic

[Isadora]

Hello,

I dont understand what is the purpose of using two-step PCR?

and when should we use it?


Thank you

[pcrman]

I assume you mean two step RT-PCR. In two-step PCR, you first convert your RNA to cDNA by RT reaction, then you can use the resulted cDNA to amplify many genes to assess their expression. While in one-step PCR, the RT and PCR reaction happen in the same tube, the converted cDNA can only be used to amplify one gene and used once.

[Trof]

I also know "two-step PCR" that means cycling two temperatures (denaturing and annealing) instead of three. It's used in real-time PCR in probe assays, it's quicker I think, but the annealing time has to be increased.
That's probably not the case, but if..

Edited by Trof, 19 January 2012 - 09:59 AM.