The TopoTA Cloning® Kits are designed for rapid cloning of a Taq polymerase amplified PCR product, without using restriction enzymes and time-consuming ligation reactions. The kits contain all reagents necessary to perform efficient and rapid cloning: pCR®2.1-Topo® vector, salt solution (final 200 mM NaCl, 10 mM MgCl
2), dH
2O, transformation reagents (E.coli DH5-T1, SOC media and a control vector) and sequencing primer (M13) for verification of the cloned insert, as well as the reagents for the initial PCR reaction (dNTPs, 10x buffer, control DNA).
TA cloning is a fast cloning procedure since the Topo-vector is provided linearized and contains T-overhangs at the 3’ end and Topoisomerase I covalently attached. Therefore, Taq-amplified PCR products with 3’A-overhangs can be easily inserted. Attention must be paid when choosing the PCR enzyme, since some of the proofreading Taq-polymerase mixtures might not produce 3’A overhangs. If needed, A residues can be added in an additional reaction described in the manual of the kit.
The reaction is set up at room temperature with 0.5 -4 µl of the PCR amplified DNA, 1 µl salt solution, 1 µl of the pCR®2.1-Topo® vector and dH2O to a final volume of 6 µl. 5 min is the recommended incubation time; this can be extended to 30 min to obtain more colonies for larger PCR products (>1kb). 2 ul of the cloned product is then ready for transformation into E. coli DH5-T1 provided as ready-to-use aliquots in single tubes.
I used different PCR products of unknown genomic DNA sequences which were amplified with a polymerase mixture containing proofreading Taq-polymerase and standard Taq polymerase, which added the A overhangs on the 3’ ends of the PCR product. To avoid having any genomic DNA contaminants, I gel-purified the PCR product and extracted the DNA from the gel using a Qiagen gel-extraction kit before cloning. From some PCR reactions, the amount of gel-purified DNA was low, so that I used 4 µl for cloning, which worked as well as lower volumes with higher DNA concentrations. Using 2 different amounts of DNA for initial cloning reactions, I realized that using less DNA for cloning into the pCR®2.1-Topo® resulted in more colonies. In subsequent reactions, I used only one DNA concentration in order to lower the cost of the procedure. The amount of vector provided in the kit was only enough for 19 cloning reactions (instead of the 20 advertised), possibly due to multiple pipetting steps.
I routinely incubated for 8-10 min since my products were up to 900 kb. When plating 2 different volumes of bacteria (10 and 50 µl) on LB-Plates with kanamycin (30 or 50 µg), colonies were small after an overnight (12 h) incubation time; incubation for 15-16 h was sometimes necessary to obtain a good size colony. The lower Kanamycin concentration (30 µg) resulted in more colonies. My cloning efficiency was slightly lower than the company advertised: between 80 and 100%, depending on the size of the inserted PCR product. I obtained up to 80% efficiency for larger products (900 kb). Since I did not observe dark blue colonies when plating bacteria on X-gal, I used regular LB-plates and screened miniprepped DNA with restriction enzyme digests for the right insertion product. Since the sequencing primer sites (M13 primer) are very close to the DNA insertion site (115/96 bases before/after), insert sequences close to 900 bp could be read reliably.
The kit is very well developed and provides a fast and reliable method for cloning and transformation of PCR products into bacteria. It is easy to use even for people with little experience in molecular biology and cloning. For some specific applications (e.g. larger products (>1kb) or PCR products which require amplification with a proofreading enzyme (this might reduce the A overhangs), the kit might be less efficient, but still sufficient for successful cloning and sequencing.