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University of Wisconsin–Madison

DNA Sequencing Facility

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 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.
Our general guidelines for sequencing primer design:
17-20 mers
40-60% GC
No more than 3 G’s or 3C’s in a row
G or C at 3’ end
Make the last 6bp on the 3’ end as unique as possible to the entire template.
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.
Unless otherwise specified, all data and reagents distributed by the University of Wisconsin Biotechnology Center DNA Sequencing Facility are intended for research purposes only. They are not intended nor certified for diagnostic or clinical use.  Clinical services are provided through our collaboration with the UW Collaborative Genomics Core.