The cloning of a PCR-generated product, whether it has been amplified from cDNA or genomic DNA, is an essential step for many biologists. Traditionally, cloning was achieved by the “restriction enzyme cutback and ligation” method, where primers were designed with restriction sites to allow for the generation of cohesive overhangs on an amplified product for subsequent ligation into a plasmid with similarly digested ends. This method is problematic due to the need of some restriction enzymes to have a template with numerous nucleotides in addition to those in the recognition sequence at the 5’/3’ end in order to digest the DNA. Another approach was to utilize the terminal transferase activity of Taq DNA polymerase which adds a single 3´-A overhang to each end of a PCR product. A linearized plasmid would be generated with a 5’-T overhang to mimic a cohesive end digest and allow ligation and subsequent cloning of the 3’-A-tagged PCR product. Invitrogen further developed on this premise by utilizing the enzyme DNA topoisomerase I (hence TOPO) to rapidly ligate the PCR product into a linearized plasmid. This works by using a linearized plasmid with DNA topoisomerase I covalently bound to each 3´ phosphate. Since the development of the first generic cloning and sequencing TOPO® TA Cloning™ Kit, Invitrogen has expanded the system with a multitude of vectors for various downstream clone-ready functions (e.g. mammalian expression). However, a lot of users still stick with the original TOPO® TA Cloning™ Kit with the pCR2.1 plasmid.
I have used the TOPO® TA Cloning™ Kit for many years for a multitude of applications with great success. Application have included the cloning of small PCR products for sequencing (100-300 bp) and larger PCR fragments generated from genomic DNA or cDNA (500-5000 bp). The kit comes with enough reagents to perform 20-40 cloning reactions, sequencing primers, a control PCR template and primers to test your PCR system, competent bacteria in “one-shot” format (100 ul aliquots for each reaction) and enough SOC medium for bacterial growth.
The manual is clearly written and very easy-to-follow. Before cloning a DNA product, a PCR amplification strategy must be chosen. If Taq DNA polymerase is to be used for the PCR, only the final extension time of the PCR needs to be altered to allow adequate A-tailing of PCR products. If you want to use Pfu to amplify your PCR product, an extra step must be included after the PCR by adding Taq DNA polymerase and performing a further extension reaction (as PFU enzymes do not add A’s to PCR products). An alternative is to use a PCR enzyme mix of a proofreading enzyme and Taq. This information and protocols for each option are included in the manual and I would recommend following these guidelines.
Once the PCR product is generated and purified, the protocol and experiment is very quick and easy to perform. At room temperature, a reaction is set up consisting of a small volume of the purified PCR product, TOPO vector (which has enzyme conjugated to it) and salt solution (provided). Invitrogen recommends allowing this reaction to proceed for 5 mins, but I found if you have products >500 bp, leaving the reaction for 15 -30 mins improves cloning efficiency. Although Invitrogen doesn’t recommend using the kit for products over 3 kb (a TOPO® XL kit is available for this), I’ve had success with multiple PCR products over 3 kb. After adding your reaction to the tube of bacteria, a typical heat-shock and growth phase of the bacteria is performed before plating overnight on LB-agar. Another good feature of the pCR2.1 TOPO cloning vector is the ability to screen for successful cloning by blue/white colonies. Clones can then be picked and further processed.
In conclusion, this is a very useful kit for cloning PCR products which Invitrogen has continued to improve since its release. The recent development of Mach-T1 bacteria (which can be ordered with the kit instead of TOP10 bacteria), allows for 8 hour growth of colonies and hence the potential isolation of cloned PCR products in 1 day. I recommend this kit as a general cloning system for PCR products.