Choosing the right primers is a crucial factor in successful automated Sanger DNA sequencing. Here are a few things to keep in mind when choosing primers for Sanger sequencing:
Length of the primer should be in the range of 18 and 24 bases.
The primer must have a GC content of about 45-55%.
The primer must have a GC-lock on the 3' end.
The melting temperature of the primer should be higher than 50°C but lower than 65°C.
The primer should be located at least 50-60 bases upstream of your sequence of interest.
The primer should not include poly base regions.
·Check primer for specificity in annealing to template.
Avoid primers with secondary structures or the potential to self-hybridize.