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.