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.
- 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.
- 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.
- Select Cloning > TA Cloning.
- 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).
- Select pBAD-TOPO from the Vector drop-down menu.
- 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.