In this video, we'll create and design primers that flank a promoter and incorporate a restriction site for BamHI.
Create a primer from your sequence
Open a DNA sequence, go to your "Sequence Map" view, select a region, and right click. From the dropdown, select "Create Primer", and select the direction you'd like.
A "Design Primer" tab will appear that displays other parameters to assist you in designing your primer.
Design, verify, and save your primer
Follow these steps to effectively design, verify, and save your primers.
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Add the BamHI restriction site sequences to your primer by searching for it in the Cut Site dropdown.
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Add more bases by typing directly or copy-and-pasting into the Bases box.
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Change the overhang length by using the arrows or typing in a number. (Any bases that appear in gray are part of the overhang).
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Check for secondary structures in the Verify section and specify the temperature if you wish. Make sure all changes in energy are above -9.0 kcal.
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Click on the Gibbs Free Energy (ΔG) values to view homodimer and monomer structures.
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Check the melting temperature (Tm) and GC content. You can click on the wrench icon next to Tm to modify other thermodynamic parameters for priming.
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Input a name for your primer and choose the color for it to appear on your sequence.
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Choose an appropriate Project or Folder to save your primer and hit "Save Primer".
Create primer pairs
If you would like to design a second primer, to create a primer pair, please follow the instructions below.
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Don't save your primer and select the "Primer Pair" from the dropdown menu.
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Make sure that you are in "Split Workspace" with the tabs "Sequence Map" and "Design Primer" both available.
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Drag and left click over a region of your sequence and choose "Set from Selection"
You can now follow the previous same steps to further design, verify, and save your second primer.