Directional TOPO Cloning in CodonCode Aligner

Directional TOPO cloning is a one-step molecular cloning technique that allows for the efficient insertion of PCR products into plasmid vectors with a defined orientation, using Topoisomerase I-mediated ligation. This method helps ensure correct gene expression by controlling the direction in which the insert is cloned. The direction is established by designing the insert so that it contains a CACC overhang at the 5’ end, which is usually introduced by PCR with a modified primer. The linearized vector contains a matching 5’ GTGG overhang, that is ligated by Topoisomerase I to the insert in the correct orientation defined by the overhangs.
In CodonCode Aligner, you can plan and simulate directional TOPO cloning virtually — from primer design to in silico ligation — to ensure accuracy before stepping into to the lab. Aligner’s intuitive interface and smart cloning tools help streamline your workflow, minimize cloning errors, and increase experimental success.

Directional TOPO cloning interface in CodonCode Aligner, showing the PCR fragment selection with CACC overhang

How Directional TOPO Cloning Works in CodonCode Aligner

Find out how to use the directional TOPO cloning tool in CodonCode Aligner.

CodonCode Aligner menu, showing how to start the TOPO cloning wizard

Start Cloning Wizard

Start the virtual cloning tool "Directional TOPO Cloning..." available from the "Tools" menu. The wizard will guide you through the cloning process.

Plasmid map, showing a selected fragment for cloning

Select Fragment

Select the PCR fragment. Clicking on an annotation selects the corresponding bases. The 'CACC' overhang is automatically added.

TOPO cloning software, showing how to choose the TOPO cloning vector

Choose Vector

Choose the vector sequence. Common commercial linearized vectors for directional TOPO cloning are listed and pre-loaded, or you can import or create your own linearized vector.

Design Primers Automatically

Design primers with a single click. The primers for directional TOPO cloning are designed by Aligner to include the required 'CACC' overhang.

TOPO cloning simulation, showing the cloning product with CACC/GTGG overhang ligation

Simulate Ligation

Simulate the ligation of fragment and vector. Aligner will warn about problems like mismatching overhangs, or incorrect primers.

Cloning product showing the plasmid map and insert orientation.

View Cloning Product

View the cloning product to preview plasmid map, annotations, feature directions, and insert location.

Verifying the reading frame with amino acid translation and start codon

Verify Clone

Check correct overhangs, features, or enzyme cut sites, and verify the reading frame.

Create and export primers and constructs directly in the cloning wizard

Export and Document

Create sequences for all of your constructs for documentation, and save and export your primers and cloning sequences.

Best Practices and Tips for Directional TOPO Cloning in CodonCode Aligner

Directional TOPO cloning is a quick and effective method for inserting PCR products into vectors, but accuracy in both experimental design and in silico simulation is key to success. CodonCode Aligner helps you plan and verify your constructs virtually — but following a few best practices can ensure the process is as smooth and error-free as possible.

Verify Overhangs
CodonCode Aligner checks your vector to verify it has the needed overhangs. Aligner also automatically generates the forward primer for your PCR fragment with the necessary 'CACC' extension. Commercial linearized vectors with the correct overhangs come pre-loaded and are available durig virtual cloning. If you want to use your own vector, you can add the required overhang manually. Aligner will warn you if your vector does not have the correct overhang.
No 5' Phosphates On Primers
Primers for TOPO cloning should not have 5´ phosphates, as Topoisomerase I requires a free hydroxyl group for ligation. If your primers are phosphorylated, you will need to remove the phosphate group before using them for TOPO cloning. TA-cloning works for phosphorylated products, but not TOPO cloning.
Increase Incubation Time For Larger Inserts
For most applications, 5 minutes at room temperature is sufficient. However, for larger inserts or cloning a collection of PCR products, increasing the reaction time can lead to better results.
Fragment:Vector Ratio
Too much or too little PCR product can significantly decrease the efficiency of TOPO cloning. Thermo Fisher recommends a 0.5:1 to 2:1 molar ratio of PCR product:TOPO vector.

Explore Other Cloning Methods and Features in CodonCode Aligner

CodonCode Aligner supports a variety of molecular cloning techniques beyond directional TOPO cloning. Have a look at these pages to learn how Aligner can help you with your molecular cloning:

Gibson Assembly
Seamless DNA assembly without the need for restriction enzymes — ideal for multi-fragment cloning and scarless constructs.
Restriction Cloning
Simulate restriction enzyme digests, ligation, and insert orientation with support for hundreds of enzymes.
TA Cloning
Learn how to simulate TA cloning and verify insert orientation before going to the bench.
Blunt-End PCR Cloning
Plan and test blunt-end ligation of PCR products and vectors, with annotation and map preview features.
Molecular Cloning
Overview of virtual molecular cloning methods in CodonCode Aligner.
Primer Design
Design primers using Primer3. Adjust parameters like primer characteristics and reaction conditions to create PCR, cloning, and sequencing primers that specifically match your workflow.
Virtual Gels and RFLP Analysis
Use virtual gels to verify and plan any gels you use in the lab. You can look at restriction maps and virtual gels for single sequences, or even compare multiple sequences (for example your cloning product in both directions) using the RFLP analysis tool in Aligner.