How do I simulate inverse PCR with a circular plasmid?
Select the Region to Remove
To select the region to be removed, e.g., by clicking on a feature.
Invert the Selection
To invert the selected region, click Edit → Invert Selection.
Open the PCR Dialog
To open the PCR dialog, click Actions → PCR... .
Choose the Primers
To automatically choose the primers, type the desired Tm and click Choose Primers.
Phosphorylate the Primers
In the PCR dialog, review the primer names and phosphorylation state. Add a phosphate group to at least one primer by clicking one of the 5' Phosphorylated check boxes.
Verify the Polymerase Selection
Verify that the Polymerase: menu shows the Creates Blunt Ends selection.
Name the Amplified Fragment
When you are ready to simulate inverse PCR, type the name of the amplified fragment, then click PCR.
View the Fragment
The linear fragment will be shown in a new window.
Circularize the Fragment
To circularize the fragment, click Actions → Circularize... .
Type the name of the circularized vector and then click Circularize.
View the Product
The circular product will be shown in a new window.
Review the History
To review the steps performed, switch to History view.