How do I simulate TA/GC cloning in SnapGene?
TA cloning is used to clone PCR products. It takes advantage of sticky end overhangs created by addition of 3′ A nucleotides due to the terminal transferase activity of Taq polymerase (and some other polymerases). These overhangs enable annealing and ligation into a linearized vector with complementary 3′ T overhangs. GC cloning is similar, and is based on the finding that Taq actually adds a mixture of 3′ A and 3′ G overhangs.
SnapGene provides a library of commonly used TA and GC cloning vectors.
See our video on TA/GC cloning for further information.
Open a File containing the Insert Sequence
Open a sequence file. If the region to be cloned is annotated as a feature then click on the feature to select it.
If the region is not annotated then switch to Sequence view and select the specific region to be amplified.
Start the TA or GC Cloning Tool
To perform TA cloning, click Actions → TA or GC Cloning → TA cloning.
To perform GC cloning, click Actions → TA or GC Cloning → GC cloning.
In this lesson we will simulate TA cloning. All steps described also apply for GC cloning.
The cloning tool opens showing the Insert panel. The selected region in the foremost open sequence file will be automatically selected as the Insert for cloning.
If you wish to change the insert source, click the "Source of Insert" dropdown menu and choose an alternative source file, then select the insert region.
Use the Dropdown to set the polymerase used for PCR. Normally you would use a polymerase that creates 3' overhangs †.
† If you choose to perform PCR with a polymerase that creates blunt ends then you will need to simulate addition of 3' overhangs after PCR, see the section below).
Choose a Vector
Switch to the "Linearized Vector" panel and select a vector from the list of "Commercial Linearized Vectors".
If you wish to use a "custom linearized" vector then use the dropdown menu to select a custom vector from your sequence collection.
Design PCR Primers
Switch to the "Product" panel and click Choose PCR Primers, set the target Tm for new primers, then click Choose Primers. New PCR primers will be added to the sequence.
Switch to the "Insert" panel and name the new primers (optional).
Enter an appropriate name for the new plasmid product.
A warning will always be shown for TA or GC Cloning: TA cloning is a non-directional procedure as the insert can potentially ligate to the vector in forward or reverse orientation.
Simulate Cloning in the Forward and Reverse Orientations
Uncheck the option "and close this window"
Set an appropriate name for the first new product plasmid.
Click Clone to create the new plasmid sequence.
The new unsaved product sequence will open. Click File → Save to save the new sequence to an appropriate location on your computer.
Switch back to the TA cloning window, switch the "Orientation of Insert", give the new product an appropriate name, then click Clone.
A new unsaved "reverse" product sequence will be created. Click File → Save to save the sequence to an appropriate location on your computer.
View History of the New Files to See the Simulated Cloning Steps
Select a "product" file and switch to "History" view to see all steps simulated during the TA or GC cloning procedure.
Adding Overhangs After PCR
If you follow the above procedure but choose to "Create Blunt Ends" (for example, if choosing to amplify PCR product using a polymerase with higher fidelity than Taq polymerase) then you will need to simulate addition of overhangs post-PCR.
After designing primers, click Add 3' A overhangs to Insert to simulate addition of A overhangs (A-Tailing) by treatment of the PCR product with Taq polymerase. Then click Clone.
An additional "Edit DNA Ends" step representing addition of the A overhangs to the PCR product will be present in the product History view.
Order Primers to Start TA or GC Cloning
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.