Deciding the annealing temperature for PCR...? - annealing temperature calc
Why is one of the objectives of using an annealing temperature (Tc) of about 5O use under primer Tm lower there too?
Monday, February 15, 2010
Annealing Temperature Calc Deciding The Annealing Temperature For PCR...?
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Think about the basics of PCR, the denaturation of the template, primer annealing, and after the enlargement.
When the annealing temperature. is too high, one or more of the primers can not glow. Therefore, we recommend going 5 gr. below the lowest Tm of the first pair.
My suggestion is to try the primer with TM, always the same, about 40-60% GC, and with a program touchdown. To ensure that your primer annealing at optimum temperature and loyalty will not decrease with a very low MC.
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