Support
User Guide PDF
The user guide is still being constructed. If you have a question about a topic that is not covered here, please contact us.

Mutagenesis

How do I simulate 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.

Create the Bottom-Strand Primer

If a bottom-strand primer is desired, click Reverse Complement.

If desired, edit the primer name.

The bottom-strand primer will be displayed below the double-stranded template sequence.

Click Add Primer to Template. Then click Close.

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.

Copyright © 2018 GSL Biotech LLC | Site Map | Privacy | Legal Disclaimers   Subscribe to Our Newsletter