what we should take note when design primer?

Answer 1

Ah, my favorite question! I assume you mean DNA oligonucleotides for use in PCR amplification? Use the A/T = 2 degrees + G/C = 4 degrees rule. Design each primer so that the Tms are identical. I like to design a Tm of 62 degrees and then anneal at 58 degrees. Make sure the last nucleotide of the primer (at the 3' end) is a C or a G.

That's it. Now you can do perfect PCR too.