Multiple Primer Design with Primer 3

This is the public gateway to a multiple primer design implementation of the Primer3 algorithm in the Bellen Lab, Baylor College of Medicine. Please read the Copyright Notice and Disclaimer of the original version, (c) Whitehead Institute.
This implementation allows you to automatically design tiled primer pairs with custom length and overlap for up to 50 kB fragments. By using this program you agree to the linked terms and conditions.
Also available: Original Primer3 (c) Whitehead Institute (runs locally in the Bellen Lab at Baylor College, Houston, TX)

Start at bp in the sequence.
Enter Overlap Value:


  1. Don't worry about the Overlap value. 25 is just fine; the actual overlap is bigger and determined by fields on the next form. Also don't worry about the Start at value. You will normally just want to start at the beginning - and that's 1. So, just click the start button. Sometimes, when it takes the program too long to find a primer pair, the flow of output is interrupted in the middle of the batch. That's when you use the Start at value to resume.
  2. Dont worry about all the fields in the next Primer3 form. All fields are pre-filled. All you do is the following: Paste the sequence of max. 50kb into the big textbox. The pre-set values produce primer pairs every 500bp with an average and optimal length of 600bp. Hence, the total overlap is on average 100bp. These 100 bp are the sum of the 25bp overlap we defined earlier plus +/- 75bp which are the difference between the 525bp fragment minimum size and the max. If you get a lot of output where primer pairs were not found, try first to reduce the input in the target field from 50,525 to 50,500. This increases the minumum overlap by another 25bp and the loop runs in 475bp steps instead of 500bp steps.
  3. Press Pick Primers. That's it. Now it starts running ... and running .. and running. Every primer pair takes between a few seconds and up to 2 minutes, depending on how hard the primers are to find for the Primer3 algorithm. This web server has been configured to transmit a stream of output without giving up for hours if need be - so just leave the output window open. In rare cases the Primer3 output will simply lack any primer data. In that case look at the position and design custom primers for the lacking piece. If this happens too often - just increase overlap as suggested above.
  4. The output is produced consecutively: Every time a new primer pair has been calculated it's added to the list. You may copy and paste the lines starting with LEFT PRIMER and RIGHT PRIMER into separate files. That means, you should have two editors or word windows open, one with the list of all left primers, one with the list of all right primers.

Note: Primer3 is copyrighted by The Whitehead Institute. This is an adaptation for multiple primer design by P. Robin Hiesinger ( Website maintained by Karen Schulze ( in the Bellen Lab.