How's everyone doing?
Just a quick one. I read that the annealing temperature for PCR primers is about 5'C from the melting temperature.
I have two degenerate primers (modified a little from different sources). Both are 27-mer primers but I got two different melting temperature range for them both: (I realize that different programs give different values)
Forward primer: 57.5 to 65.9
Reverse primer: 77.0 to 79.7
I came upon one Japanese paper using 65'C, 2 mins for annealing and elongation.
65'C at 2 mins seemed too much for the forward primer, no?
And i'm sure primer design isn't that simple. There are other things like dimer, delta G, and stuff. Can anyone provide me a link to programs that is really reliable in analyzing primers?
Thanks in advance, people!
Edited by dreamchaser_jc, 19 April 2009 - 06:36 AM.