How do I simulate Q5® Site Directed mutagenesis in SnapGene?
SnapGene does not have a dedicated tool for design of appropriate primers for Q5 site directed mutagenesis.
However, you can use the procedure described below to simulate Q5® Site Directed mutagenesis in SnapGene.
The NEB Q5® Site Directed mutagenesis kit utilises Q5 polymerase. This polymerase is fused to a processivity-enhancing dsDNA binding domain that stabilises a primer-template complex. This means the optimal annealing temperature for Q5 mediated PCR is 6-12°C higher than that predicted for annealing in the presence a regular polymerase.
We recommend use of the NEB BaseChanger online tool for design of primers suitable for NEB Q5® Site Directed mutagenesis see – https://nebasechanger.neb.com
Define the Mutation
Open your circular plasmid sequence and identify the site/region for mutagenesis, taking note of the region coordinates if performing a deletion, or the codon number for mutation. In the example above, we wish to mutate GFP codon 65 from serine to threonine, S65T.
Click on the CDS feature to select the full CDS sequence and click Copy to copy the sequence to the clipboard.
Design Mutagenic Primers
In a browser go to https://nebasechanger.neb.com, paste the copied sequence into the field provided, set the input mode, enter the desired mutation, then click Build Primers.
Consult the documentation at https://nebasechanger.neb.com for details on correct use of the NEB BaseChanger primer design tools.
Click Download All Primers to save the newly designed primers to an appropriate location on your computer as a tab-delimited text file.
Add the Primers to your Target Sequence
Switch back to SnapGene.
Click Primers → Import Primers → Import Primers from a List....
Click Browse to load the newly created tab-delimited primer file.
Uncheck the option to "Require a perfect match".
Click OK to import the primers onto your sequence.
Confirm a single match is found on your plasmid sequence for both primers, then click Add 2 Primers.
Verify the two primers bind in the appropriate location on your template sequence. Click Save.
Perform the PCR and Mutagenesis
Q5® Site Directed mutagenesis in SnapGene has main two steps,
1) Q5-mediated PCR, followed by
2) Incubation in KLD enzyme mix for K) phosphorylation of PCR product by kinase, L) ligation of the the phosphorylated PCR product, and D) digestion of any wild-type template by the restriction enzyme DpnI.
The following steps simulate PCR, phosphorylation and and ligation (circularization of the PCR product) to create the expected product of a Q5® mutagenesis procedure.
Click Actions → PCR.
Use the Primer: dropdowns to set the new primers as the mutagenic forward and reverse primers for PCR.
Ensure the "Polymerase:" is set to "Create blunt ends".
Click PCR to generate the Q5® PCR product.
View the PCR product file and click Actions → Circularize.
Check to options to phosphorylate at least one strand of the PCR product, then click Phosphorylate.
Enter an appropriate name for your final Q5 mutagenesis product and click Circularize.
Click Save to save the new sequence to an appropriate location on your computer.
Switch to Sequence view, click on the minimap to navigate to the mutagenesis location and confirm the mutagenesis is correct.
Create a New Feature Representing the Mutated CDS
The above procedure splits the original CDS into two separate features. Follow the procedure below to create a new "mutant" feature.
Click on the left CDS feature to select it (1), the SHIFT-click on the right feature (2) to select the full range of the original CDS feature.
Click Features → Add Feature.
Enter an appropriate name for the new Feature, set the Type: to "CDS", check the option to "Translate this feature in Sequence view".
If required, set a color for the new feature.
If required, add product information to the feature.
Click OK to add the new feature to the sequence.
Switch to Features view, select the old Features, and hit the "delete" key to delete the old features.
Click Save to save your Q5® mutagenesis product.
Switch to Map view to view the new correctly annotated mutated CDS feature.