We are going to create primers that flank the DsRed2 gene and incorporate the restriction sites for BbvCI and PacI.
Create a primer from your sequence
- Open a sequence map, select a region, and right click.
- From the dropdown, select Create primer, and select the direction. The Design Primer tab will appear on the right and your selected sequence will be in the Bases box. Any bases that appear in gray are part of the overhang, whereas bases in green indicate the binding region.
Design and verify your primer
Here are some parameters you can use in the design and verification process
- Add restriction site sequences to your primer from using the Cut Site dropdown
- Add in a more bases by typing directly into the Bases box
- Change the overhang length by using the arrows in the Overhang box.
- Check for secondary structures in the Verify section. Make sure all changes in energy are above -9.0 kcal.
- Check Tm and GC content, and modify the buffer parameters (click the wrench icon)
Create primer pairs
To design your second primer, select the Single Primer drop down menu and click on Primer Pair.
Repeat the steps you took to make your first primer.
- Check for secondary structures by going to the Verify section and making sure all changes in energy are above -9.0 kcal.
- Save your primers by going to the Save section. Choose to save in the My Training folder and click Save Primer Pair.