How do I simulate PCR amplification and directional cloning of a DNA fragment into a directional TOPO® Vector?
SnapGene provides a library of all commercially available TOPO® vector sequences, ready for use in SnapGene.
If required, SnapGene will automatically design appropriate insert-specific primers for directional TOPO® cloning.
Open the TOPO Directional Cloning Dialog
Click Actions → TOPO® Cloning → Directional TOPO® Cloning... to open the "Directional TOPO® Cloning" window.
Choose the Insert
If you open and view a sequence file prior to clicking Actions → TOPO® Cloning → Directional TOPO® Cloning..., then the open sequence will automatically be set as the "Source of Insert".
Select the Insert tab and click the Source of Insert menu to choose the "Insert" file from the list (if recently viewed) or select "Browse" to locate the sequence file on your computer.
Select the region of the selected sequence that will be amplified for directional TOPO® cloning. Click on the Feature in Map or Sequence view to select it, or switch to Sequence view to select a precise region.
Click the "Orientation of insert" buttons to set the desired orientation of the insert.
Choose the Vector
Switch to the "Linearized Vector" tab and select the vector you will be using from the list of "Commercial Linearized" directional TOPO® vectors.
Design Primers for the Insert
SnapGene will design appropriate forward and reverse primers to amplify the insert. The forward primer will include the "5'-CACC" motif required to ensure directional TOPO® cloning.
Switch to the Product tab and click Choose PCR Primers... to design new insert-specific PCR primers.
Confirm the correct region will be amplified, set the desired primer Tm, then click Choose Primers.
If your insert already has the 5' "CACC" motif needed for directional TOPO cloning then you may not need to design PCR primers. In this case, in Insert view, set the "Source of Insert" and choose to "Use directly as the template".
Note that the "Use directly as the template" option is only available if the "5-CACC" motif is present on your sequence.
Confirm the Predicted Insert Sequence and Ligation Product
New primers will be designed to amplify the insert sequence. The forward primer will include a 5' "CACC" motif that confers directionality to the TOPO®-mediated ligation of insert to vector.
In the Product tab, switch to Sequence view and confirm the simulated ligation is in the correct orientation and in-frame with vector-based reading frames.
If you need to insert nucleotides into the forward or reverse primers to ensure in-frame fusion then switch back to the Insert panel and manually edit the primer sequences.
Enter a name for the new "product" sequence.
Name the New Primers
Switch to the Insert panel. Edit the names of the newly designed primers.
Create the Vector/Insert Product Sequence
The SnapGene Directional TOPO® cloning tool simulates PCR amplification and topoisomerase-mediated ligation steps. You can choose to output a separate file representing the intermediate PCR product.
In the "Create product" field, enter a name for the product.
Note that by default SnapGene will output only the final circular insert/vector product.
Optional: You can check the option to "Make File" for the "Insert" to create a separate new file for the intermediate PCR product. Enter an appropriate name for the Insert PCR product file.
Once you have selected and named the product/s to be created, click the Clone button to simulate the insert PCR and TOPO®-mediated ligation steps. The new "product" file(s) will open in separate new windows.
For each new "product" file, click the Save button on the top toolbar to save the file to an appropriate location.
Order the Primers
You can export the new primers to a delimited text file to use for "cut and paste" ordering
Click the Save button dropdown in the top toolbar and choose to Export Primer Data... in a delimited text format that can then be used for primer ordering.