I always wonder do we have to calculate the Tm for the whole primer, or only for the sequence complementary to the gene of interest? the TM for the blue regions are 50 and 64C respectively, but for the whole primer are 67 and 72C.
CAGACGTCTCGTATAGGGACCAAACAGAGAATCTGTGAG (TATA and GGG are introduced)
Expected size: 3700 b
My PCR doesn't work. I use pfu.
Edited by Curtis, 18 September 2011 - 08:43 PM.