TA & TOPO Cloning Calculator
Clone a Taq-amplified PCR product into a T-vector or TOPO vector and get the resulting plasmid sequence.
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Both a vector and an insert sequence are required.
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What it does
TA cloning exploits the terminal transferase activity of Taq polymerase, which adds a single 3′ A to most PCR products. A linear vector with complementary 3′ T overhangs can then be ligated to it without any restriction sites. TOPO cloning replaces ligation with topoisomerase I, which is already covalently bound to the vector ends.
How it works
In TA mode the product is built as T + insert + A + vector, reproducing the single-base additions at each junction. In TOPO mode the vector's CCCTT overhang pairs with the insert's CACC 5′ extension and GTGG 3′ end, so the product is CACC + insert + GTGG + vector. Both leave a small junction footprint you may want to account for in a coding sequence.
Worked example
A 1.0 kb Taq product cloned into a 3.0 kb T-vector gives a 4.0 kb plasmid plus the two junction bases. Switching to TOPO mode adds the four-base CACC/GTGG pair instead, which is what determines the reading frame in a TOPO expression system.
When to use it
Use it for quick cloning of screening PCR products, for TA cloning of non-proofread amplicons, and to confirm what a TOPO vector contributes to the final construct before you order one.
FAQ
- Why does Taq add an A overhang?
- Taq polymerase has terminal transferase activity and adds a single non-templated dA to the 3′ end of most products. Proofreading polymerases do not, so their products must be A-tailed first.
- Can I TA-clone a product from a proofreading enzyme?
- Not directly. Blunt-ended products need an A-tailing step with Taq and dATP, or you should switch to blunt-end or seamless cloning.
- What is the difference between TA and TOPO?
- TA uses DNA ligase to join the A/T overhangs and takes minutes to hours; TOPO uses vector-bound topoisomerase I and completes in about five minutes, but needs specific vector systems.
- Which orientation will the insert go in?
- TA cloning is not directional — the insert goes in both orientations roughly equally, so screen colonies by colony PCR or digest. TOPO directional vectors avoid this.