Primers and annealing temperature - (Apr/19/2009 )

Hey guys,

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!

dreamchaser_jc on Apr 20 2009, 12:24 AM said:

Unfortunately, no. Because the Tm is affected by local sequence, as well as the straightforward classical H-bonds between strands, every equation to calculate Tm will have a degree of compromise. One thing is for certain, you need to redesign the primers to bring the Tms closer together.
do the different equations you have used give one primer with a consistently lower Tm than the other one? Can you extend that one a few bases? Or can you shorten the high Tm primer? What are you planning on doing with the products?

swanny on Apr 20 2009, 08:09 AM said:

Thanks swanny. Yeah, calculations gave different Tms but the Forward is always lower than the Reverse. I'd see if I could add or remove some bases

F 5'-CCSCCSTGGATCAAYAAGTWYTAYATC

R 5'-SAGCCASGCSGTCCARTCSGGCCACCA

If not, I'd just redesign. The primers are from highly conserved regions from a number of bacteria. Just an early step to fish for part of the gene and then design a more specific primer with the goal of mapping the genes. Thanks again swanny.