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Design forward and reverse sequencing primers using our free PrimerQuest Tool. After opening the tool, click Show Custom Design Parameters, and choose the forward or reverse sequencing parameter set.
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.
We recommend using our free OligoAnalyzer® Tool to assist in primer design. Primers should be between 18–30 nt long with an optimal length of 20–25 nt. They should have a G-C content between 40-60% and a Tm between 55–65°C. Avoid primers that form strong self-dimers or hairpins, and those that are 90% homologous to other regions within your sample. We recommend running a BLAST search to check for sequence homology (this can also be done in OligoAnalyzer).