AAT Bioquest

How do I choose primers for Sanger sequencing?

Posted July 11, 2022


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.
Additional resources

In-silico Designing and Testing of Primers for Sanger Genome Sequencing of Dengue Virus Types of Asian Origin

MagaDye™ 4 Color Sanger Sequencing Terminator Kit