Primer Design

General guidelines

1. The lengths of primers should be 17-25 bases.
2. GC% should be 40-60%
3. Melting temperature (Tm) should be approximately 50%
4. Usually the 3' base should be a G or C.
5. Ensure that primers cannot hybridize to themselves or each other with a stable structure, especially one that forms continuous base pairing at the 3' end of a primer.

Tools for primer design

1. OligoAnalyzer from IDT
   Web form that calculates melting temperatures as well as self- and cross-hybridization.
2. PrimerSelect from DNASTAR
   Software program for designing primers.

Primers for cloning or sequence construction

1. If adding a restriction site to the end of the PCR product, remember to add ~6 extra base pairs for the restriction enzyme.