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How to choose the parameters (Tm, cycli) for RT-PCR - (Jan/04/2010 )


I`m trying to design a RT-PCR from scratch. I`ve designed my primers and
have their Tm. But if I`m going to test them in RT-PCR how do I know which program I have to use?

I`m talking about annealling temperature, number of cycli...??
Are there just standard parameters you use and you just fill in your Tm for your primers?



you need to run gradient to determine the exact annealing temp.

you can also buy kits for RT-PCR from various companies. the protocols are in the kits or you can even find them online. there are two types of RT-PCR; one-step and two-step.

it would be better if you could have a look at protocol-online's main page for more info.