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

Use the following parameters in the design and verification process.

  • Add restriction site sequences to your primer from using the Cut Site dropdown.
  • Add 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 the melting temperature (Tm) and GC content. Modify the buffer parameters by clicking the wrench icon next to Tm.

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 a specific folder in which to save your primers and click Save Primer Pair.

What's next? 

After reviewing this tutorial you can learn how to attach saved primers, design primers with the primer wizard, and run PCR in silico.

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