PCR optimization: PCR vs qPCR - (Jun/08/2012 )

The correct way to start a qPCR is to find the better Tm of the primer, isn´t it?

So, we perform a gradient of conventional PCRs to discover that. But the concentrations of reagents of a conventional PCR is different from those of qPCR and the concentrations of reagents change the Tm of the primer.

My question is: how can I labor with these differences? Is this relevant? Or the same Tm of the primer in a conventional PCR will be the same in a qPCR?

I make sure my qPCRs all work at the same temperature and in the same qPCR master mix. There's no point to optimize for PCR with one polymerase and buffer and then change to another polymerase and buffer.

Are you mean that you have been used the same Tm for all your primers? This is strange because each primer have it´s own Tm, isn´t?

I meant that I use a single annealing/extension temperature: 60C. I design all of my primers using PrimerBlast and leave the primer melting temperatures set to the default Min: 57, Opt: 60, Max: 63 with a maximum Tm difference of 3 between the forward and reverse primers. When a primer pair gives an ugly melting curve, I just design new primers.

First I performed the qPCR reaction in 60C, but the qPCR efficiency was poor 82%. I think the melt curve is ok.

I think it could be the tm of primer, but I dont know. What do you think?


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