How do I simulate Blunt TOPO® Cloning?
SnapGene provides a library of all commercially available TOPO® vector sequences, ready for use in SnapGene.
If required, SnapGene will automatically design appropriate insert fragment-specific primers for directional TOPO® cloning.
To watch a video on how to simulate TOPO cloning in SnapGene see this link.
Open the Blunt TOPO® Cloning Dialog
Click Actions → TOPO® Cloning →Blunt TOPO® Cloning... to open the "Blunt TOPO® Cloning" window.
Choose the Fragment
If you open and view a sequence file prior to clicking Actions → TOPO® Cloning →Blunt TOPO® Cloning..., then the open sequence will automatically be set as the "Source of Insert".
Otherwise, select the Insert tab and click 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 sequence that will be used in the Blunt TOPO® reaction.
In Map view, click on a feature to select the feature or switch to Sequence view to select a specific region.
Choose the Vector
Switch to the "Linearized Vector" panel and select the vector you will be using from the list of "Commercial Linearized" Blunt TOPO® vectors.
Design Primers for the Insert
Switch to the Product tab and click Choose PCR Primers... to design new insert-specific PCR primers.
Set the desired primer Tm, then click Choose Primers.
Confirm the Predicted Insert Sequence and Fusion Product
In the Product tab, switch to Sequence view and confirm the simulated fusion 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 create or remove an in-frame fusion then switch back to the Insert tab 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.
Create the Insert/Vector Product Sequences
SnapGene assumes you will perform PCR with a DNA polymerase that does not have template-independent terminal transferase activity (e.g. Taq polymerase), resulting in a blunt end PCR product.
Blunt TOPO® cloning is non-directional, so in practice two insert/vector products can be generated. You can choose to have Snapgene output two separate files representing ligation of the insert in the forward and reverse (flipped) directions.
To create "Product" sequences, switch to the "Product" tab.
By default SnapGene will output only the "forward" version of the insert/vector product.
Optional: To make intermediate products do the following:
- Check the option to "Make File" for the "Insert" to create a separate file for the intermediate PCR product. Enter an appropriate unique name for the Insert.
- Check the option to "Make File" for the "Alternative Product with Flipped Insert" to create a separate file with the insert in the opposite orientation. Enter an appropriate unique name for the alternative product.
Once you have selected and named all primers and products to be created, click the Clone button.
All selected "products" files will be created and opened in 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.