After the wet lab team spent hours designing primers by consulting with various online tools and using some good old guess and check, our dry lab team came to the rescue and designed a Primer Design calculator. After inputting a sequence you need a primer for, it will provide a list of potential primers sorted by Tm, and will also test for all common restriction sites and indicate their presence to the user! Feel free to test it out - maybe you can help improve it!
The table of thermodynamic values used and the method for melting temperature calculation are found in the paper [SantaLucia JR (1998) "A unified view of polymer, dumbbell and oligonucleotide DNA nearest-neighbor thermodynamics", Proc Natl Acad Sci 95:1460-65 http://dx.doi.org/10.1073/pnas.95.4.1460.
Simply take your sequence, and insert two percentage signs as shown (in red), indicating where you would like the program to test for primers of varying lengths. Copy paste your sequence into the variable 'Sequence' in the code below, and specify which lengths of primer you would like. We did not have the time to develop a graphical user interface as we would've liked, but you can still try out our algorithm in the interactive code box below. This code assumes qPCR, DNA primers;
Oligo Conc 0.2 µM
Na+ Conc 50 mM
Mg++ Conc 3 mM
dNTPs Conc 0.8 mM