Design forward and reverse sequencing primers using our free PrimerQuest® tool
(under the SciTools® Tab
). After opening the tool, click "Show Custom Design Parameters", and choose the forward or reverse sequencing parameter set. Primer Position: The best sequencing data is typically found 80-150 bp out from the primer; it is difficult to get good base calls <50 bp and >300 bp from the primer. Primer Design: Primers should be between 18-30 nt long with an optimal length of 20-25 nt; have a G-C content between 40-60%; and a Tm
between 55-65°C. Avoid primers that form strong self-dimers or hairpins. Use our free OligoAnalyzer® tool
(also found under the SciTools Menu) to check for such structures. Avoid primers which are 90% homologous to other regions within your sample. We recommend running a BLAST search to check for sequence homology; this can be done from within the OligoAnalyzer software.