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2-step or 3-step real time PCR - question about real time PCR (Nov/04/2009 )

Hi, right now I am doing a protocol in real time PCR using the Biorad Icycler machine using a 3-step PCR before data collection and melt curve analysis. I've read somewhere that a 2-step PCR (denaturation then annealing) is enough. Can anyone please explain why this is so? Thank you.

-pop09-

Normally RT PCR or Q-PCR is done for a small template.. ideally less than 150 bp.. and the polymerase can entend this number during the ramp time irself.. so a different step for extension is not generally needed in real time!!!
Hope this helps..
any other explanations are welcome!!

-Pradeep Iyer-

Pradeep Iyer on Nov 4 2009, 10:11 PM said:

Normally RT PCR or Q-PCR is done for a small template.. ideally less than 150 bp.. and the polymerase can entend this number during the ramp time irself.. so a different step for extension is not generally needed in real time!!!
Hope this helps..
any other explanations are welcome!!

-pop09-

Thank you so much for that!

-pop09-

I may be wrong on this, but I thought one of the reasons you could do a two-step PCR with qPCR is because of the taq enzyme that is typically used in pre-made master mixes. Whereas the typical taq enzyme used in end-point PCR works best at 72C, the taq used in the qPCR master mixes works at 60C, the same temperature which is typically used for primer annealing. Thus you only need to denature at one temp then anneal/extend at another temp....please correct me if I'm wrong.

-Mighty Mouse-

Mighty Mouse on Nov 6 2009, 09:56 AM said:

I may be wrong on this, but I thought one of the reasons you could do a two-step PCR with qPCR is because of the taq enzyme that is typically used in pre-made master mixes. Whereas the typical taq enzyme used in end-point PCR works best at 72C, the taq used in the qPCR master mixes works at 60C, the same temperature which is typically used for primer annealing. Thus you only need to denature at one temp then anneal/extend at another temp....please correct me if I'm wrong.



i ve never heard of that but seems to be a good explanation too!!! all i know about premixes is that they are blocked.. the Taq and they need to be activated before the amplification (95 degreees 5 min). And also it is not necessary to use only master mixes.. taq and sybr for example can be used separately too.. as far as the 60 degrees one is concerned.. i l check and let u know...

-Pradeep Iyer-

Hi MM.. i got this from the Eurogentec site about the Taq they use in their master mixes

HotGoldStar is a recombinant Hotstart activated-Taq DNA polymerase.

It is purified from an E. coli strain, which carries a Thermus species DNA polymerase overproducing plasmid.
It is a modified Taq polymerase, which completely lacks any activity below 74 C that avoids non-specific priming at low temperature. HotGoldStar requires a thermal activation of 10 minutes at 95 C to reach maximal initial activity. During the PCR the rest of its activity is released. It is heat-degraded in a much lower rate as commonly used Taq DNA polymerase.

Hotstart activity
No primer dimers
No non-specific products
TA cloning

Hope its clearer now!!! Good u raised the question.. i serarched :rolleyes:

-Pradeep Iyer-