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Automated DNA Sequencing - Primers

Good primer design is one of the key factors in obtaining sound sequencing data. Using either online tools or stand-alone programs, please design your primers according to the recommended properties below. Shown below is a small list of freely available tools for primer design. Please DO NOT add two PCR primers into your reaction. One sequencing reaction, one primer please!

Primer Design Tools

Recommended primer properties

  • Primer concentration = 5µM.
  • 18-26 bases long.
  • Optimum Tm is great than 50 oC but less than 65oC.
  • Around 50% GC.
  • Avoid primers with secondary structures or the potential to self-hybridize.
  • Avoid runs of a single base (e.g. 3 to 4 repeats of the same base)

UMass User can order from Fisher Scientific for free shipping - Click the  Find the solution that meets your requirements-from oligo design to custom RNA products including siRNA, miRNA and many more.

Free Primers Provided by Umass Genomics Resource Lab