Primer design principles

Posted onAuthorBaradwaj G Ravi

Primer design principles

 

  • The Tm should be around 52-56°C. Tm can be calculated from multiple online tools.​​ (normally Benching is used to calculate the Tm)

  • The Tm differences between​​ a pair of primers should not exceed 1°C.

  • It’s preferable to have a G/C at the 3’ end of your primer for better extension

  • Avoid tandem repeats like “AAAA” if possible. It’s hard to synthesize these sequences.

  • Don’t forget to use the reverse complement sequence for your reverse primer!

  • For Gibson cloning, add 20~25 nt additional sequence that overlaps the vector to the 5’ of the primer. For traditional cloning, add respective restriction digestion sites to the primer, as well as 2~4 additional nucleotides to​​ facilitate restriction enzyme binding. Note, these additional sequences are not included when calculating Tm.

  • Be mindful about your primer length. Any oligo exceeds 60nt can be very expensive. Try to stay under 60nt if possible.