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How to choose the parameters (Tm, cycli) for RT-PCR

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#1 Blackeyed



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Posted 04 January 2010 - 01:10 AM


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?


#2 Curtis


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Posted 07 January 2010 - 01:42 AM

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.

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