Primer Considerations

There are dozens of internet sites that have primer design programs, hints, and explanations. We have only looked at a few of these, but the following URLs are some sites that had somewhat handy easy to use primer design programs. Also is a summation of the criteria we use when designing oligos for sequencing. Unlike PCR, it’s pretty hard to design a primer that doesn’t work for sequencing.

http://alces.med.umn.edu/rawprimer.html Primer design

http://www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi Primer design

http://genome-www2.stanford.edu/cgi-bin/SGD/web-primer Primer design (best one for sequencing primers)

Our general guidelines for sequencing primer design:

18-20 mers

40-60% GC

No more than 3 G’s or 3C’s in a row

G or C at 3’ end

That’s it! We’ve had pretty good luck with those simple rules—haven’t really had to worry about hairpins or primer dimers for sequencing. If you find your primer gives peaks under peaks, try the following:

Have it resynthesized (degraded primers give peaks under peaks)

Recheck the concentration

Raise the annealing temperature, add DMSO to 5%

Check your predicted sequence for repeat regions

Unlike for standard PCR, it’s difficult to make primers that don’t work for sequencing. So don’t spend too much time agonizing over the perfect primer sequence. If the region you need doesn’t correspond to a favored composition, give it a shot anyway—chances are it will work fine.