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 neighboring bases and takes the primer and sodium concentrations into account.
Check your primers
- Paste your primers, one per line, into PCR Primer Stats.
- Set the primer and sodium concentrations of your reaction.
- 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.
Checking one primer
Take 5′-AGCGGATAACAATTTCACACAGG-3′. It is 23 bases long, with 10 G or C, so the GC content is 10 ÷ 23 = 43%, inside the 40 to 60% range. It ends in GG, a GC clamp, with three G or C in the last five bases, the usual maximum. The simple 2 + 4 rule gives 2 × 13 + 4 × 10 = 66 °C, which overestimates for a primer this long. A nearest-neighbor calculation with your salt conditions is more reliable.
Annealing temperature
A common starting point is about 3 to 5 °C below the lower Tm of the pair. If unsure, run a gradient PCR across several temperatures and pick the one that gives a single clean band.
Adding a restriction site
To clone the product, add the enzyme's site to the 5′ end of the primer, with 4 to 6 extra bases before it so the enzyme can cut near the end. Work out the Tm from the part that binds the template only, since the tail does not bind in the first cycles.
Common problems
- No product. Lower the annealing temperature, check the primer sequences against the template, and confirm the template is there.
- Several bands. Raise the annealing temperature, and look for other places the primers could bind.
- A faint band below 100 bp. This is usually primer dimer, the primers joined to each other. Redesign to avoid complementary 3′ ends, or lower the primer concentration.