Deciding the annealing temperature for PCR...?

Question

Why should one aim at using an annealing temperature (Ta) about 5oC below the lowest Tm of ther pair of primers to be used?

Answer 1

Think about the basics of PCR- denaturation of the template, annealing of the primers, and then extension.
If the annealing temp. is too high, one or more of the primers may not anneal. That's why it is suggested to go 5 deg. below the lowest Tm of the primer pair.
My suggestion is to try to always order primers with equal Tm, GC% around 40-60, and use a touchdown program. This will make sure your primers anneal at the ideal temperature and your fidelity doesn't decrease with too low of a Tm.

Answer 2

so some distance as i understand melting temp is while 0.5 the DNA is uncoiled and the rest remains bonded. think of of it as a zip which u zipped down halfway, this factor while the zipper is halfway down is the melting temp. Annealing temp is the choice. Its the factor while the separated DNA varieties decrease back bonds and get caught at the same time back. desire that helps :)

Answer 3

Try this website..
http://www.mcb.uct.ac.za/manual/pcroptim.htm#Annealing