How do I design primers for site-directed mutagenesis?
Identify the Mutagenesis Region
First, identify the site you want to change. About 15-18 bases of a mutagenic primer should anneal to the template on each side of the mutagenesis site.
Click to place the cursor 15-18 bp upstream of the mutagenesis site, then drag to create a selection that ends 15-18 bp downstream of the mutagenesis site.
Open the Add Primer Dialog
To open the Add Primer dialog, Click Primers → Add Primer... .
Create the Top-Strand Primer
In the Add Primer dialog, click Top Strand.
If desired, edit the primer name.
Choose the Mutagenesis Site
Choose the mutagenesis site in the text box. For a deletion or replacement, select the relevant bases. For an insertion, click to place a cursor between the flanking bases.
Modify the Top-Strand Primer by Deletion
If the mutagenesis involves a deletion, the site of the deletion will be marked by red and blue color gradients in the text box and by an elevated placement in the primer display.
Modify the Top-Strand Primer by Insertion
If the mutagenesis involves an insertion, the inserted bases will be marked by red color in the text box and by an elevated placement in the primer display.
Modify the Top-Strand Primer by Replacement
To modify the primer, select the bases to be replaced. Type the new bases. Alternatively, click Insertions to switch the tab, and then proceed as described below.
Replace the selected bases as follows. Choose a codon, site, or peptide coding sequence using the menu controls, and then click the appropriate Insert button.
The inserted bases will be marked by red color in the text box and by an elevated placement in the primer display.
Add the Primer to the Template
If a bottom-strand primer will be created as well, uncheck the box labeled Close after adding primer.
Click Add Primer to Template.
Select a Mutagenic Primer
Select either a top-strand or a bottom-strand mutagenic primer.
Open the Mutagenesis Dialog
Click Actions → Mutagenesis... .
Edit the Plasmid Name
If desired, edit the name of the mutagenized plasmid, then click Mutagenize.
Show History Colors
To highlight the mutated region in the new sequence window, click the "Show history colors" button in the side toolbar. Inserted or replaced bases will be marked in red, or a deletion will be marked by red and blue color gradients.
Export the Primer Data
To export the mutagenic primers, switch to Primers view. Select the primers of interest, then click Primers → Export Selected Primer Data. See the "Export Primers" lesson for more details.