Using Primer3

Primer3 is an open source program for designing PCR primers, sequencing primers and hybridization probes. Primer3 has a huge number of customizable parameters, most of which you won’t need to worry about (i.e. the default settings for most parameters will work fine for our purposes).

Because Primer3 is open source, you can find many customized versions of it, both web interfaces and stand-alone programs. We will use Primer3Plus.

I find it is easiest to explain how to use this program using screen shots (click on the images to access the full size view). Please take some time to look over the various parameters you can modify.

The first step is to get your sequence into Primer3. You can either paste it in, or upload a file.

Primer3Plus - Main Tab

If you need the primers to be on either side of a particular portion of the target sequence, use one of the fields below the sequence input field of the Main tab.

If you already know what primers you want to use, and are simply wanting a nice print out showing the primer on your target sequence, paste the primer sequences into the appropriate fields. In this case, you don’t need to worry about other settings as you are forcing Primer3 to use these primers regardless.

To customize settings for primer length, GC content etc. and to set the product size range, go to the General Settings tab.

Primer3Plus - General Tab

To customize the 3’ GC content of the primers, go to the Advanced Settings tab.

Primer3Plus - Advanced Tab
Do not change any other parameters unless you are confident that you know what the parameter actual does. From within Primer3Plus you can check what a parameter does by mousing over or clicking the associated hyperlinks.

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