Primer Design Tutorial

Do you need to design a primer pair specific for your sequence of interest?

You are in the right place! This tutorial expains how to use Primer3Plus to generate primers for any coding sequence.

Before you start designing your primers find your sequences:

• Please, look at the “Search gene” and/or “Sequence similarity search” tutorial to learn how to find your sequence of interest.
• Once you find it, click on the name and then on the “Sequences” tab on the left column.
• Under the mRNA sequence the “Design Primer for …” link is available.

Design your primers:

• The “Main” menu, found on Primer3Plus website will already have your sequence of interest in the sequence box.
  • The tools on the bottom allow you to include or exclude part of the sequence from the primer design and to tell to the system if you already have one primer (left/forward or right/reverse) and you need to design only the other one.
• Understanding the “General settings” menu:
  • The primers can be customized in many different ways, for example, the optimal primer length, the optimal primer amplification product length, the optimal melting temperature (Tm), and optimal Guanine and Cytosine % (GC%).
    • The primer size is usually 20 base pairs (bp) (min 18 bp and max 27 bp).
    • The primer Tm is usually set around 58°C for cloning purpose (min 56°C and max 60°C); but it is usually higher if the primers are used for other purpose.
    • The GC% of a primer is usually 50% (min 20% and max 80%).
    • All the other characteristics are pretty much standard.
    • Expert users can use the “Advance settings”, “Internal oligo”, “Penalty weights” and “ Sequence quality” menus to customize the primer features.
    • More information and details about designing primers can be found in the link that are in the top right corner.
• Design your primers:
  • Click “Pick Primers”.
  • The optimal primer pair designed using your required characteristics is shown at the top of the page.
  • For this primer pair, the position in the sequence, the length, the Tm, and the GC% are reported.
  • Also reported for the primer pair is the size of the amplified product and any possible alignment between one primer F and one primer R.
  • Following the overview of the primer pair, the entire sequence is showed and the optimal primer pair is highlighted.
  • After the sequence, another 4 primer pairs are reported together with all the mentioned characteristics.
• Ordering your primers:
  • Click on “Send to Primer3Manager” under the primer pair that you selected.
  • In this window you can click “Check!” to recall all the features of each selected primer and “BLAST!” to be driven directly to the NCBI BLAST tool, in order to check that your primer is able to bind only to your sequence of interest or also checking that your primers will not give you non-specific amplifications.
  • When you click “Order selected primer” you will have a list of primer names and primer sequences that you can copy and paste and send to the e-mail address of the company that will provide you the primer.