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Two-Step PCR

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2 replies to this topic

#1 Isadora



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Posted 18 January 2012 - 01:37 PM


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

and when should we use it?

Thank you

#2 pcrman



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Posted 18 January 2012 - 08:15 PM

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.

#3 Trof


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Posted 19 January 2012 - 09:58 AM

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.

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