Simulating PCR
It is possible to simulate PCR and automatically have Photo51 design primers for your planned experiment. Start by navigating to the “PCR” tab in the top banner, here find and select the “PCR” button, this will open the PCR modal box.
In the PCR modal box you have the option to “Create primers from selection” this automatically design primers that will amplify the region you have selected. Regions can be selected by clicking and dragging across the plasmid map or the sequence, alternatively you can select click a features to select the region it covers or use shift to select a range. The other option available is to “Create primers to linearize at cursor” this option automatically designs primers that create a linear sequence, select a single point in the plasmid and primers will be created that amplify the whole sequence.
You can set the type of polymerase (blunt ends or 3’ A/G overhangs) and the target annealing temperature as parameters for your primer design. Once you have selected the correct region or placed the cursor at the desired linearization point, use the corresponding button at the top left to automatically generate the primers, these will be displayed under Primer 1 and Primer 2 below the set parameters. Note that if you have already created primers you can either search them by name by typing their respective name into the primer name box next to “Primer 1 / 2” or your can simply paste your desired primer sequence in 5’ to 3’ orientation in the primer sequence box.
For each primer the default names are set as PCR.For and PCR.Rev for Primer 1 and Primer 2 respectively. You can change the names by clicking into the box. Below the name the primer sequence is displayed in 5’ to 3’ orientation. Here it is possible to add any custom base pairs into your primer, these will be shown in green. Below the primer sequence you can select to add a 5’ phosphorylation modification to the via the “Select modification” dropdown. Below this you will see the current length and annealing temperature of the primer you have created. Finally the last option is to add restriction sites to your primer, via the “Select enzyme” dropdown; first select the desired restriction enzyme type then select how many extra upstream bases to add to the 5’ by default the manufacturer’s recommendation is used, once ready select “Add site to primer” and you will see the restriction site added to your primer sequence.
Once you have finished your primer design, you can preview the PCR result via the “PCR result” button above your plasmid preview, this will show you what the final product of your PCR will look like. If the preview looks correct you can navigate to the bottom right of the modal and select “Next”.
This will open the PCR summary view here there are three sections, input a name for your product, optionally select a location to save the file (otherwise it will simply be placed in the root) and lastly a summary of the final primers that were used for PCR. To create and save the product select save at the bottom right of the box and your product file will be saved and opened for you to view.
PCR Mutagenesis
THIS SECTION NEEDS TO BE WRITTEN
CANNOT NOW BECAUSE ISSUE:
- Cannot remove following warning even though I have done everything correctly: “To enable mutagenesis, add lowercase bases in Primer 1/2 within the selected region (mismatches required).”