Invitrogen’s TA Cloning Kit (Dual Promoter)

The TA cloning kit from Invitrogen proves a highly efficient method of cloning a PCR product into a plasmid vector in one step. We have used the TA cloning kit extensively in our lab for cloning various genes and gene fragments. The principle behind the TA cloning kit is quite simple. Taq polymerase, the most common polymerase used in PCR, adds a single deoxyadenosine to the 3’ end of PCR products. In the TA kit the linearized pCRII vector is provided with corresponding 3’ deoxythymidine overhangs. This allows the efficient insertion of the PCR product into the pCRII vector.

The main advantage of TA cloning kit is that it is quick, convenient to use and saves time. It does not require design of primers containing restriction sites. If the PCR product is pure (we purify our PCR products using Geneclean) the cloning efficiency is high. The kit is supplied with all the reagents required in sufficient quantities and you do not have purchase extra reagents from outside. The manual with the kit is excellent and provides detail protocols and troubleshooting. If you are using thermostable polymerases like Pfu, which do not leave 3’ A overhangs, a protocol is supplied by which you add them at the end of the PCR reaction. The pCRII vector in the kit is provided with a kanamycin resistance gene, which is especially useful in cloning fragments PCR amplified from plasmids with ampicillin resistance. The insert can be sequenced using the T7 or the Sp6 sites provided in the vector.

The pCRII vector contains Sp6 and T7 promoters upstream and downstream to the multiple cloning site, which allows in vitro transcription of the insert. Although we have cloned many genes into pCRII, we have not had much luck with the in vitro transcription and translation of the gene products. In fact, often we sub-clone the fragment into pcDNA3 after TA cloning to allow efficient transcription-translation of the products. This may be at least, in part, due to the nature of the insert and not due to the kit itself. Nevertheless, I would highly recommend the TA cloning kit as a valuable tool for molecular biology research.

Piyali Dasgupta, Ph.D.
Post-Doctoral Fellow
Moffit Research Center