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Primer Design
General
- Primers should generally be 20–22 nt long with 10–12 Gs and Cs
- Remember to use the reverse compliment for your reverse primer
Sequencing
- Design one forward primer for every ~500 bp
- You won't get good sequence where your primer sits, so design your first primer to be a little upstream from your sequence
- You only need one reverse primer
Cloning
- Check for restriction sites within your target before adding them to your primers
- Add a "CGC" to the ends of your primers
Gene Deletion
- Check for restriction sites within your target before adding them to your primers
- Add a "CGC" to the ends of your primers
- Clone ~500 bp upstream and downstream of the gene you're intending to delete and include ~20 bp of the target gene
- For your internal primers, give them 10-12 bp homology with each other for Overlap-extension PCR
Primer Hydration
- Find the nmoles on the tube (e.g. 24.65 nmoles)
- Multiply by ten (e.g. 24.65 × 10 = 246.5)
- Take that number and add that many μL of TE buffer (e.g. add 240 to 250 μL)
- This will give you 100× primers (i.e. 100 µM)