How do I simulate Blunt TOPO® Cloning?
Open the Blunt TOPO® Cloning Dialog
Click Actions → TOPO® Cloning →Blunt TOPO® Cloning... to open the "Blunt TOPO® Cloning" window.
Choose the Insert
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".
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 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
SnapGene provides a library of 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.
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.
Confirm the correct region will be amplified, set the desired primer Tm, then click Choose Primers.
Create the Insert/Vector Product Sequences
Blunt TOPO® cloning is non-directional, so in practice two insert/vector 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" tab.
By default SnapGene will output only the "forward" version of the insert/vector product.
- In the "Create product" field, enter a unique name for the forward product.
- Optional: 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.
- Optional: 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 the products to be created, click the Clone button. The new "Product" file(s) will open in new separate 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.