What were the factors determine higher annealing temperature for molecular markers like RAPD, ISSR and SSR,s? How can we identify the annealing temperature? Is it depends up on no of bands in a gel or by a particular band having our desired product size ? Whether annealing temperature have any complementary relation with the GC content, melting temperature and the number of bases in the molecular marker?
basically anealing temp defined on the primers fragments but i don,t believe that this temp may be adjusted because its neat to denaturation
Yes, annealing temperature can be influenced by the the percentage of CG in the primers. I think 80 C seem to be very high tm. You have to recalculate your tm.
For markers like RAPD, annealing temperature is generally around 38 to 40 degree celsius. Mainly it depends upon GC content. For others like ISSR and SSR you may try gradient temperature of Tm+/- 5 degrees. Tm (melting temperature) may be available from the company from where you got your primers synthesized. Or else, it may also be calculated using the formula or even the below link will help you.you.http://www6.appliedbiosystems.com/support/techtools/calc/
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