Which program should I use - (Dec/13/2007 )
The Tm of my primers are about 60 degree, and the length of the DNA fragment is bout 350 bp. How long should I set the denaturing, annealing and elongation time for the real-time pcr. Could you give me some help? Thanks.
at the top of this page, you'll see a tab called "protocol"
there are links to more information than you could ever want about PCR in general, and about real-time specifically as well
the main problem I foresee is that your amplicon is very long for efficient copying