How do I simulate PCR amplification?
Open the Template Sequence
Open the sequence that will be the template for PCR.
In Map view or Sequence view, select the region for amplification.
Start the PCR Tool
To open the PCR dialog, click Actions → PCR....
Create PCR Primers
If the region for amplification is selected prior to running the tool, the tool will automatically open the "Choose Primers" dialog.
If no sequence was open or selected prior to running the PCR tool, then select the "Template" sequence file via the dropdown, select the region for amplification, then click Choose PCR Primers....
Set the desired target Tm for the primers then click Choose Primers.
Specify Details and Create the PCR Product
In the PCR dialog:
- Add primer names.
- Set the phosphorylation state (note: oligonucleotides are usually synthesized and supplied without 5' phosphates).
- Manually add primer 5' extensions if required.
- Use the "Polymerase:" dropdown to choose the type of ends for the amplified fragment.
- Name the PCR Product.
Once all details are specified click PCR to create a new "product" file.
View and Save the Product
The product will be shown in a new tab or window.
Click File → Save to save the product sequence to an appropriate location on your computer.
PCR with Preexisting Primers
To perform PCR using preexisting primers on a sequence, open sequence file, click to select the forward primer, then Shift-click to select the reverse primer plus the intervening sequence, then click Actions → PCR....
Review the PCR History
To view the product history, switch to History view.
A list of PCR primers and their sequences can be obtained by clicking the primer names. The primer sequences can then be copied and pasted into a text file.
