PlasmidLab / Guides / PCR primer design

PCR primer design: length, Tm and 3′ ends

A primer pair has to bind your template, bind nothing else, and not bind each other. Most of the usual rules of thumb come from one of those three requirements.

The numbers most primers follow

• Length 18 to 25 bases. Shorter primers lose specificity; longer ones rarely help.
• Tm around 55 to 65 °C, calculated with a nearest-neighbour model that takes your salt and primer concentrations into account. A Tm from the old 2 °C per A or T, 4 °C per G or C rule is too rough for anything but the shortest primers.
• The pair within about 5 °C of each other, so one primer does not sit idle while the other anneals.
• GC content 40 to 60%.
• A G or C at the 3′ end. The last few bases decide whether the polymerase extends, so they matter more than the rest.
• No run longer than four identical bases, which slips on repeats.
• No 3′ complementarity between the two primers, or between a primer and itself. That is how primer dimers start, and once they start they out-compete your product.

These are starting points, not rules. A primer that breaks one of them and works is fine; a primer that follows all of them and amplifies two bands is not.

A tail does not count in the first cycles

When you add something to the 5′ end, a restriction site, a Gibson overlap, an attB site or a tag, only the 3′ annealing part base-pairs with the template in the first two cycles. Set the annealing temperature from that part alone. From the third cycle the template carries the tail too, and the whole primer anneals.

A restriction site at the end of a product also needs a few spare bases beyond it, because most enzymes cut poorly at the very end of a fragment. The restriction cloning guide covers how many.

Check specificity

A pair that looks perfect can still prime somewhere else in the genome. Select the primers and choose Analyze > Search NCBI with Selected Primers to check them against the database. The sequence you selected is sent to NCBI for that search, so read the privacy page first if the sequence is confidential.

In PlasmidLab

Annotate > New Primer… creates a primer on the open sequence and shows its properties. Annotate > Primer Library… keeps the primers you already own, so you can look for one before you order another. Clone > PCR… simulates the reaction and makes the product, and Annotate > Walk Me Through Primers explains the steps inside the app.