In Silico TA Cloning

TA cloning is a simple and efficient method for inserting PCR products with 3′-A overhangs into linearized vectors with 3′-T overhangs. It is a widely used cloning method that does not require restriction enzymes. PCR products for TA cloning are amplified using Taq DNA polymerases, which adds a single A overhang to the 3' end of product. This allows for efficient ligation of the PCR product into a vector with complementary T overhangs. CodonCode Aligner brings this method into the digital lab, allowing you to plan and simulate TA cloning workflows with precision and ease.

How TA Cloning Works in CodonCode Aligner

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

Start Cloning Wizard

Start the virtual TA cloning wizard. It will guide you through the cloning process step by step.

Select Insert

Select your PCR product (insert). Simply click on a feature annotation to select the whole sequence for it. A overhangs are automatically added.

Choose Vector

Select the target vector sequence. Use your own custom vector, or choose from one of the common vectors for TA cloning that come pre-loaded in CodonCode Aligner.

Automatically Design Primers

Automatically design suitable primers for your experiment. You can manually edit the primers, for example to include spacers.

Simulate Ligation

Simulate the ligation. Potential problems like mismatching overhangs are flagged by the software automatically.

View Cloning Product

View the cloning product to preview insert orientation, plasmid structure, and annotated features.

Verify Clone

Check correct A/T overhangs and verify the reading frames. Automatically create the alternate cloning product and compare with virtual gels or alignments.

Export and Document

Save your constructs for future reference, and export your primers directly for ordering.

To learn more about TA cloning in CodonCode Aligner, look at the TA cloning tutorial.

Best Practices and Tips for TA Cloning in CodonCode Aligner

TA cloning is an 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. By following a few best practices you can ensure the process is as smooth and error-free as possible.

• Check Insert Orientation
  Use Aligner’s preview tools to confirm that your insert is in the correct direction — TA cloning is non-directional, so verifying this virtually can prevent unwelcome surprises. You can use Aligner's restriction mapping tool to preview a fragment gel map that can help you verify the correct insert direction using unique restriction enzymes.
• Negative Control
  Include a negative vector-only control in your ligation to help identify background from self-ligated vectors.
• Use freshly prepared PCR products
  A-overhangs can degrade over time and reduce ligation efficiency, so using recent PCR products can increase your ligation success.
• Verify Overhangs and Features
  CodonCode Aligner checks your vector to verify it has the needed overhang. If you do need to edit your vector, you can add the required overhang before starting your virtual cloning experiment.
• Leverage Plasmid Map Visualization
  After ligation simulation, review the annotated map to ensure correct insertion, reading frame, and absence of unwanted restriction sites.
• Saving Your Constructs
  Use descriptive names when saving virtual constructs — this helps track changes, share designs, and revisit earlier versions if needed. Aligner allows you to create new samples from all the constructs in your cloning experiment: primers, cloning product, linearized vector, and insert.
• Ordering Primers
  You can direcly export the primers used during virtual cloning to fasta files for ordering.

Explore Other Cloning Methods and Features in CodonCode Aligner

CodonCode Aligner supports a variety of molecular cloning techniques beyond TA cloning. Explore these additional pages to learn how Aligner helps you design, simulate, and verify different cloning workflows:

• 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.
• Directional TOPO Cloning
  Learn how to simulate directional TOPO 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 your insert direction. 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.