How do I simulate TA 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-specific primers for directional TOPO® cloning.
Open the TOPO® TA Cloning Dialog
Click Actions → TOPO® Cloning → TOPO® TA Cloning... to open the "TOPO® TA Cloning" dialog.
Choose the Insert
Select the Insert tab and use 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 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 (Taq polymerase) with template-independent terminal transferase activity resulting in the addition of overhanging adenine (A) to the 3' ends of the PCR product.
Switch to the Product tab and click Choose PCR Primers... to set the desired melting temperature (Tm) for new PCR primers.
Confirm the correct region will be amplified, set the desired primer Tm, then click Choose Primers.
Confirm the Predicted Insert Sequence and Fusion Product
New primers will be designed to amplify the insert sequence. The simulated PCR amplified insert will include 3'-terminal R (A or G) overhangs.
In the Product tab, switch to Sequence view and confirm the simulated fusion 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 an in-frame fusion then switch back to the Insert tab and manually edit the primer sequences.
Enter a name for the new "product" sequence.
Rename the New Primers
Switch back to the Insert tab. Edit the names of the newly designed primers.
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.
In the "Create product" field, enter a name for the product.
You can check the option to "Make File" for the "Insert" 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 the products to be created, click the Clone button. The new "product" file/s will open in separate new windows.
If you choose to output all alternative and intermediate products then 3 new (unsaved) files will be created.
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
Select a product sequence, then 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.