Inverse PCR

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.