TOPO Cloning Tutorial: Blunt Cloning

Topoisomerase-mediated Blunt cloning is very similar to Topoisomerase-mediated TA cloning and in Geneious there is virtually no difference in how TA- and Blunt-vectors & inserts are treated. The mechanism of the topoisomerase is the same as for TA cloning as illustrated below.

Picture taken from Zero Blunt™ manual, Thermo Fisher Scientific.

To highlight the power of the TOPO cloning operation we're going to show that in Geneious you can set any sequence to be treated as a TOPO site. Select the blunt Vector and navigate/zoom to the annotated multiple cloning site.

This vector does not have a standard TOPO CCCTTAAGGG motif. We will define and use a non-standard motif, AGGCCT, which is flanked by two EcoRI sites (coordinates 3172 - 3184). This TOPO site won't be detected automatically, so we will have to add a TOPO annotation.

Select the bases AGGCCT (coordinates 3177 - 3182) and click on 'Add Annotation'. Enter any name you like, and type TOPO into the Type: field. The Direction of the annotation does not matter, it will be ignored as TA & Blunt are bidirectional (a directional TOPO site will be automatically recognised). In this vector the T7 promoter is in the same direction that the insert will go in, so we do not need to reverse complement the vector.

Click Save to apply the change to the vector.

Next, select the blunt vector and human proinsulin sequences and go Cloning → TOPO Cloning). You may find that the TA settings from exercise 2 have been remembered and that a "No vectors found" error message is displayed. This is because the TA operation requires a TA-pair in the middle of the TOPO annotation. Check the Blunt Cloning option and you'll see your vector displayed, with a warning that we should recheck our annotated site. We're aware that we are using a non-standard site, so click on OK to run the reaction.

The resulting 3908 bp circular plasmid will comprise the proinsulin CDS and flanking regions ligated into the defined TOPO site of the pCR-Blunt II - TOPO vector.

Use the link below to continue with Exercise 3.

Exercise 1: Cloning into TA Vector
Exercise 3: Directional Cloning
Exercise 4: Batch Cloning

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