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. These sequences are linearized to match the linearized topoisomerase-linked vectors included with TOPO® cloning kits.
If required, SnapGene will automatically design appropriate insert-specific primers for directional TOPO® cloning.
To watch a video on how to simulate TOPO cloning in SnapGene see this link.
Open the TOPO Directional Cloning Dialog
Click Actions → TOPO® Cloning → Directional TOPO® Cloning... to open the "Directional TOPO® Cloning" window.
Choose the Fragment
If you have a file open when you start the "Topo TA Cloning" tool then the file will automatically be loaded as the "Source of Fragment".
Otherwise, select the Fragment tab and use the Source of Fragment menu to choose the fragment 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.
If required used the "Orientation of fragment" buttons to set the desired orientation when ligated into the vector.
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 fragment. 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 fragment-specific PCR primers.
Confirm the correct region will be amplified.
Set the desired primer Tm, then click Choose Primers.
If your insert fragment already has the 5' "CACC" motif needed for directional TOPO cloning then you may not need to design PCR primers. In this case, in the Fragment 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
In the Product tab, switch to Sequence view and confirm the simulated ligation is in the correct orientation and, if required, 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.
Name the New Primers and Product
Switch to the Fragment tab.
Edit to add appropriate unique names for the new primers.
Enter a name for the new "product" sequence.
The 5'-"CACC" tail added to the forward primer is annotated with an arrow.
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 products to be created, click the Clone button.
If you choose to output the intermediate PCR product then 2 new (unsaved) files will be created and opened as tabs.
For each new "product" file, click the Save button on the top toolbar to save the file to an appropriate location on your computer.
Alternatively, expand the left side panel and in the yellow "unsaved" warning, click the ellipsis and choose Save All.
Order the Primers
Export your PCR primer sequences as text for ordering from an oligonucleotide synthesis service provider. See Export Primers for more information on how to export primers.