TA Cloning

What is TA Cloning?

TA cloning is one of the simplest and most popular approaches for cloning of PCR products with the use of Taq DNA Polymerase. TA cloning is a convenient method because it is applicable to DNA fragments whose sequences are unknown (e.g. after restriction digest of complex DNA mixtures). TA cloning (also known as T-vector cloning) is widely used for cloning of single genes and the construction of DNA libraries. Since, TA cloning does not require knowledge of DNA sequences, it is especially useful for the high-throughput cloning of diverse DNA fragments. TA cloning is therefore an ideal method for high-throughput analysis of protein structure and function.

TA Cloning System

TA cloning occurs with the terminal transferase activity of a certain type of DNA polymerase e.g. Taq polymerase. The Taq polymerase enzyme adds a single, 3'-A overhang to each end to the PCR product. As a result, the PCR product can be directly cloned into a linearized cloning vector that have single base 3'-T overhangs on each end. Such vectors are called T- vectors. The PCR product with A overhang, is mixed with this vector in high proportion. The complementary overhangs of a "T" vector and the PCR product hybridize. This results in a recombinant DNA, the recombination which occurs via DNA ligase.

Assay Genie TA Cloning Products

Product Code Product Name

MORV0111


GenieClone 5 minute TA Cloning Kit

GenieClone 5 minute TA Cloning Kit is a second generation TOPO cloning kit that contains a second generation Topoisomerase, a vector containing the suicide gene ccdB and a blunt end factor. Combining with the optimal buffer, the second generation of Topoisomerase provides a highly efficient, 5 minute, one-step cloning strategy at room temperature. This product using a vector containing the suicide gene ccdB, when the insert is successfully ligated to the vector, the correct expression of ccdB is destroyed, and the host cell can grow normally, otherwise the host cell cannot grow normally, thereby achieving “zero” background. Containing a blunt end factor, 5minTM
TA/Blunt-Zero Cloning Kit is compatible with both TA clones and blunt clones.

GenieClone 5 minute TA Cloning Kit - Protocol

1. Summary of the Experimental Process

Figure A: Ta-zero Cloning
a. Add the amplification product which 3' end containing A of Taq (such as Vazyme's Taq) to 5xTA/Blunt-zero Cloning Mix, incubate at room temperature for 5 min.
b. The blunt-end factor in Mix removes the A-base at the end of the amplification product to form a blunt-ended product.
c. 5'-OH of the blunt-end product attacks the phosphate bond between the TOPO enzyme and the vector, the TOPO enzyme is released, and the vector forms a circular recombinant with the bluntended product.

Figure B: Blunt-zero Cloning
a. Amplification products (blunt ends) of high-fidelity enzymes (such as Assay Genies Fusion Ultra series) were added to 5xTA/Blunt-zero Cloning Mix and incubated at room temperature for 5 min.
b. 5'-OH of the blunt-end product attacks the phosphate bond between the TOPO enzyme and the vector, the TOPO enzyme is released, and the vector forms a circular recombinant with the bluntended product.

2. PCR Product Preparation

  • Primer requirements: the 5' end of the primer cannot be phosphorylated.
  • Enzyme selection: It is recommended to use Assay Genie Fusion series products.
  • Product requirements: Please ensure the integrity of the PCR amplification products; after the end of the amplification, the yield and quality of the product are detected by electrophoresis, if the product has only the target band, no non-specific band and primer dimers, it can be used directly, otherwise it is recommended to carry out gel recovery and purification. If the amplification template is plasmid, purification is recommended.

3. Ligation Reaction

Prepare the reaction mix.

Component Amount

GenieClone 5 minute TA Cloning Mix

1 µl

Purified PCR Product

1-4 μl

ddH2O

To 5 μl

Mix the bottom of the flick tube, collect all the liquid at the bottom of the centrifuge tube at low speed and centrifuge at room temperature (20 - 37℃) for 5 min. After the reaction was over, the tube was placed on ice.

Recommended reaction conditions:

a. The optimum amount of inserts used = [0.05 × fragment base pairs] ng;
For example, when the insert is 1000 bp, the optimum amount is [0.05×1000] ng, that is, 50 ng. Due to the wide range of compatibility of the inserts of this product, you can also use the recommended dosages in the table below:

Inserts Size Recommended Dosage

0.5 -1 kb

5 -6 ng

1 – 2 kb

60 – 110 ng

2 – 5 kb

110 – 260 ng

> 5 kb

> 260 ng

b. Reaction Temperature:
This product has high compatibility with reaction temperature, so the reaction can be performed at room temperature (20 - 37℃) (recommended by PCR instrument).

c. Reaction Time:
Let the mix react for 5 minutes.

4. Conversion

This product is compatible with many conventional competent cells (eg. DH5а competent cell; FastT1 competent cell)

*It is recommended to use Fast-T1 competent cell for subsequent transformation experiments. The cells are the fastest growing competent cells (clones can be seen 8h after plating), and the transformation efficiency is high, saving screening time.

5. Positive Clone Identification

a. PCR identification of the bacterial colony and solution:
Pick a single colony to 10 μl of ddH2O as a template; 2 × Rapid Taq Master Mix are recommended.

Reaction System:

Components Volume

2x Taq Master Mix

10 μl

M13 Primer Mix

2 μl

Bacterial Solution

2 μl

ddH2O

6 μl

Reaction Procedure:

Temperature Time & Cycles

95℃

5 min

95℃

15 sec - 35 Cycles

55℃

30 sec - 35 Cycles

72℃

60 sec/kb - 35 Cycles

72℃

10 min

b. Enzyme Digestion Analysis:
According to the experimental design, select the appropriate restriction endonuclease to identify

c. Identification of Plasmid Size:
Picking a single clone, after plasmid extraction, electrophoresis observation of plasmid size identification

d. Sequencing Analysis:
Directly pick the monoclonal sequencing identification, sequencing primers can choose M13 Forward Primer, M13 Reverse Primer or design it yourself

Attachment: Sequence Information of Vector