TOPO Cloning Tutorial: Directional Cloning

Previously we defined the orientation of our insert by reversing the insert or vector sequence. For TOPO Directional cloning this is not necessary as the insert can only be cloned in one direction due to a short (4bp) extension added to the 5'-end of the sequence, which is complementary to a vector-based TOPO extension.

Picture taken from Directional TOPO® manual, ThermoFisher Scientific.

Select the pET100/D-TOPO vector and zoom in to the Directional TOPO site annotation. You will notice that it bears a CACC in the middle of the TOPO recognition sequence.

To make our insert compatible with the vector we are going to create a primer with the CACC extension and we will design precise primers to clone the Proinsulin CDS only, without the flanking non-coding sequence. Select the Human Proinsulin sequence and click on the CDS annotation to select it. Go Primers → Design New Primers.... Set Select Task: to Design New, set Task to Precise. Check the option for Included region and ensure it shows the base numbers for the CDS (56 to 388). Set the Number of pairs to generate to 1. Expand the Characteristics panel and set Size Min: to 17 (The start of the CDS is quite GC-rich and the default Size Min of 18 will prevent forward primer design). To add the CACC extension, expand the Advanced section and click on the Fwd: none button. Choose Add: Bases and type in 'CACC'. Give the addition an appropriate Name: and Type:, for example TOPO_overhang.

Confirm all dialogs with OK, and you should see a primer pair annotated on the sequence. Click Save to make the primer annotations permanent, choosing to "deactivate links to descendants".

Now that we have a primer pair with the extension on the forward primer we're going to extract the PCR product to use in our cloning reaction. Select the Human Proinsulin sequence and go Primers → Extract PCR Product and the primers you just saved onto the sequence should be selected. Click OK.

Select the resulting sequence, called Human proinsulin PCR Product and the pET100/D-TOPO vector and go Cloning → TOPO Cloning. Select Directional with overlap: and enter CACC into the provided field.

You will notice that the Vector is only identified when the overhang sequence is entered correctly. Click OK to run the cloning operation.

Select the resulting circular product, called Human proinsulin PCR Product ligated into pET100/D-TOPO and zoom in to position 400 and you will see the ligation junction, note the CACC extension on the 54 F primer aligns with the CACC sequence in the middle of the Directional TOPO site, indicating the directional cloning operation has worked correctly.

Use the link below to continue with Exercise 4.

Exercise 1: Cloning into TA Vector
Exercise 2: Cloning into ZeroBlunt Vector
Exercise 4: Batch Cloning

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