I'm trying to design primers for analyzing SNPs via single base extension. I know I need to use a F or R primer that lines up to the SNP, but I'm trying to figure out how many base pairs to use, melting temperature and possible secondary structure. I've tried using BatchPrimer3, but it offers no primers, even when I broaden the search criteria. Does anyone have tips on how to do this, or know of a program that I can use to help design these primers? If there are tips on how to broaden the search criteria (GC content, self complementarity etc.) also, I would appreciate that.
You can use primer designing tool from pubmed: http://www.ncbi.nlm.nih.gov/tools/primer-blast/ usually it works very well. If the template is genomic DNA, you can consider using longer primers of 25 to 28 bp long. Just design 2 to 3 pairs of primers, one of those will surely work. Sometimes, the annealing temperature may need to be optimised.
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
Further suggestion to above (especially the idea of designing 2-3 primers):
I spent a few years developing high-throughput SNP technologies. When you are doing initial evaluation of a new SNP (or at least new to your lab), it can be beneficial to order and test both F and R primers for the single-base extension step. Oligos are so cheap that it's not much of an expense. The benefit is that you get confirmatory information -- the SNP data read from either direction must match (and if not, something else is going wrong). Among the issues this can help identify is whether there are nearby (possibly undiscovered) SNPs that interfere with your primers. Remember that this should always be a concern with PCR primers also, but with only 1 primer the single-base SNP assays are a bit more sensitive to this.
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