How do I simulate TA TOPO® Cloning?
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 fragment-specific primers for directional TOPO® cloning.
To watch a video on how to simulate TOPO cloning in SnapGene see this link.
Open the TOPO® TA Cloning Dialog
Click Actions → TOPO® Cloning → TOPO® TA Cloning... to open the "TOPO® TA Cloning" dialog.
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 Insert 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 TOPO® TA reaction.
In Map view, click on a feature to select the feature or switch to Sequence view to select the region.
Choose the Vector
Switch to the "Linearized Vector" tab and select the vector you will be using from the list of "commercial linearized" TOPO® TA vectors.
Design Primers for the Insert
SnapGene assumes you will perform PCR with a polymerase with template-independent terminal transferase activity (Taq polymerase) resulting in the addition of overhanging adenine (A) to the 3' ends of the PCR product.
New primers will be designed to amplify the insert fragment sequence. The simulated PCR amplified fragment will have 3'-terminal R (A or G) overhangs.
Switch to the Product tab and click Choose PCR Primers....
Set the desired melting temperature (Tm) for new PCR primers, then click Choose Primers to create appropriate 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 Vector/Insert Product Sequence
The SnapGene TOPO® TA cloning tool simulates PCR amplification and topoisomerase-mediated ligation steps. You can choose to output a separate file representing the intermediate PCR product.
TOPO® TA cloning is non-directional, so in practice two plasmid/insert products will 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 one or more "Product" sequences, switch to the Product panel.
By default SnapGene will output only the "forward" version of the insert/vector ligation.
Optional: To make intermediate products do the following:
- You can check the option to "Make File" for the "Fragment" to create a separate file for the intermediate PCR product. Enter an appropriate name for the Insert.
- You can 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 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, view the file and 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.