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Primer Selection Guidelines

The following is a list of parameters to take into account when designing primers for use under our automated sequencing conditions:

  • 20-30 nucleotides in length.
  • 50% G/C content.
  • G & C "clamps" on the 5' and 3' ends (at least a single G or C residue.)
  • Avoid multiple Thymidine residues on 3' and 5' ends.
  • Avoid primers with long runs (more than 4) of a single base.
  • Avoid primers with secondary structures or that can hybridize to form dimers or hairpins. These can be easily predicted if a primer design program is used such as primer select.
  • Melting temperature 55o-65oC.
  • Check primers for specificity in annealing to template.
  • Primers should be located at least 30-40 bases upstream of the area of interest in the sequence read.