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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:
https://primer3.ut.ee/primer3web_help.htm
2. Get the sequence of the Waxy gene, GI: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.
Which are the better results? Why?
EXERCISE 2: Make a primer pair for an SSR marker
1. Get from the NCBI the nucleotide sequence in FASTA format of:
gi:24496216
gi:3434977
gi:3434976
.
2. Align the sequences using T-Coffee at
http://www.ebi.ac.uk/t-coffee/.
3. Look for the TCTCTC repeats. Select for 360 bases which
includes the TC repeats.
4. Go to the Primer3 website.
https://primer3.ut.ee
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
https://www.idtdna.com.
Procced to the registration process, login into the site, and
into the TOOLS section choose the OligoAnalyzer Tool.
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.
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