How to Design and Check PCR Primers

What makes a good primer

• Length: 18 to 30 bases.
• GC content: 40 to 60%.
• Melting temperature: roughly 55 to 65 °C, with the two primers of a pair within about 5 °C of each other.
• 3′ end: finish on a G or C, the GC clamp, but with no more than 3 G or C in the last 5 bases.
• Avoid: runs of the same base, stretches that pair with the primer itself (hairpins and primer dimers), and stretches that pair with the other primer.

Melting temperature

The simplest formula counts 2 °C for each A or T and 4 °C for each G or C, and is only reasonable for short primers. Longer primers are better estimated with GC-based formulas, with a salt correction, or best of all with the nearest-neighbor method, which looks at each pair of neighbouring bases and takes the primer and sodium concentrations into account.

Check your primers

1. Paste your primers, one per line, into PCR Primer Stats.
2. Set the primer and sodium concentrations of your reaction.
3. Compare the Tm values and read any warnings about hairpins, self-dimers, runs or the 3′ end.

Check the product

PCR Products finds where the forward and reverse primers bind a template and gives the size and sequence of each product, so an extra binding site shows up before you run a gel. Primer Map draws a set of primers on the sequence with arrows for their direction.