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.
To watch a video on simulating TA or GC cloning in SnapGene see this link.
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
In this lesson we will simulate TA cloning. All steps described also apply for GC cloning.
To perform TA cloning, click Actions → TA or GC Cloning → TA cloning.
To perform GC cloning, click Actions → TA or GC Cloning → GC cloning.
The selected region in the foremost open sequence file will be automatically selected in the Fragment tab as the fragment for cloning.
If you wish to change the fragment source, click the "Source of Fragment" 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" tab and select a vector from the list of "Commercial Linearized Vectors".
If you wish to use a "custom linearized" vector then select the option and use the dropdown menu to select a custom vector from your sequence files.
Design PCR Primers
Switch to the "Product" panel and click Choose PCR Primers
Set the target Tm for new primers, then click Choose Primers.
Switch to the "Fragment" tab and name the new primers (optional).
Enter an appropriate name for the new plasmid product.
Click Clone to create the product.
A warning will always be shown for TA or GC Cloning: TA cloning is a non-directional procedure as the insert can ligate to the vector in either forward or reverse orientation.
Simulate Cloning in the Forward and Reverse Orientations
To create both possible products, with the fragment in opposite orientations, switch to the Product tab.
Check to option for "Alternative Product with Flipped Fragment" and give the alternative product a unique name.
Click Clone to create both product sequences.
View each product file and click File → Save to save each product sequence to an appropriate location on your computer.
View History of the New Product 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.
Click on the "TA Cloning" operation name to see History colors.
Adding Overhangs After PCR
If you follow the above procedure but choose to "Create Blunt Ends" (for example, if PCR is performed using a high fidelity DNA 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.
You will be able to proceed. click Clone to create the product.
An additional "Edit DNA Ends" step representing addition of nucleotide overhangs to the PCR product will be included 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.