The tutorial Try it! – Create and modify primers for TA cloning demonstrated how to select and modify a primer pair. This tutorial shows how to extract the PCR insert and create a virtual TA clone.

  1. If you have not already done so, follow the tutorial Try it! – Create and modify primers for TA cloning. If you closed the project since following that tutorial, use File > Open to open the file saved at the end of that tutorial.
  1. Choose Priming > Create Insert by PCR to extract the sequence of the PCR product and simulate its amplification with Taq polymerase including the addition of 3’ A overhangs to each end.

A Project window opens with a new Taq amplified insert inside the Inserts folder.

  1. Select Cloning > TA Cloning.
  1. Drag the amplified insert from the Inserts folder of the Project tab and drop it on the Fragment area of the Clone tab.

In the Clone tab’s Vector drop-down menu, note the set has been filtered to include only linear vectors, as required for TA (and TOPO) cloning. Linear vectors have the icon . To navigate through the menu items, use the vertical scroll bar (Win), or mouse wheel (Mac).

  1. Select pBAD-TOPO from the Vector drop-down menu.
  1. Click Try It and then Make It.

The forward and reverse orientation clones now appear in the Project tab in the TA Clone 1 folder.

  1. Double-click on the forward orientation clone to open it in the Sequence view. Notice that the primers and PCR product appear as features. These features can also be seen in the Features view, as well as all graphical views.

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