Primer Design 

Written by Jeffrey Detras    

EXERCISE 1: Try Primer3

1. Go to the Primer3 website: 

If you need help, you can look at the help section of Primer3 for more information regarding the parameters: 

2. Get the sequence of the waxy gene, 7798550. 

3. Use the sequence as the Source Sequence and try changing parameters and view the Primer3 Output. Explore the different options and parameters that can be used. 


EXERCISE 3: Make a primer pair for an SSR marker

1. Get the nucleotide sequence of the following at NCBI:
gi: 24496216, 3434977, 3434976 or download the Waxy_Gene_Seq.fsa.

2. Get the FASTA format of the sequences and align using T-Coffee at

3. Look for the TCTCTC repeats. Select for 360 bases which includes the TC repeats. 

4. Go to the Primer3 website.

5. Copy the selected sequence in the Source Sequence window.
6. Change the parameters:

  • Highlight the TC repeats with [TCTC...]
  • Product Size 300 300 360
  • Primer Size 18 20 22
  • Primer Tm 40 60 60
  • Primer GC% 40 50 60
  • CG Clamp 1

7. Click on the Pick Primers button. Wait for the Primer3 Output.

8. Check the suggested primer for alignment with the multiple alignment. If the primer hits a gap or a mismatch, select another primer pair that hits no gap or mismatch.

9. Check for dimerization or hairpins with OligoAnalyzer at 

10. Save the Primer3 Output as AF031162_WX_gene_primer_output.txt.

Additional Information:

The Primer Design presentation is an introduction to tools that can help you with designing primers. Tools available on the web are Primer3 and GeneFisher2 while FastPCR is a software application that you can install locally in your computer.