pGEM-T Easy Vector Systems From Promega

Promega’s pGEM-T vector systems are very dependable and versatile tools to routinely clone fragments of DNA into reliable plasmids. The pGEM-T and pGEM-T Easy plasmid vectors are essentially the same but with one important difference. The pGEM-T Easy vector has EcoRI restriction sites surrounding the proposed insert site, whereas the pGEM-T vector does not. This addition enables the ‘easy’ restriction of the plasmid for routine cloning applications, hence the name. The vectors are both just 3000 bp in linear length, which make them considerably smaller than several other vectors such as the pBIN series used in plant transformation.

I primarily use the pGEM-T and pGEM-T Easy vectors for cloning PCR products. These can be further analyzed by direct sequencing, or used for more indirect applications such as the preparation of accurate standard curves for real-time PCR experiments. Both these vectors are high copy number and have the T7 and SP6 RNA polymerase promoters flanking the multiple cloning region which lies within the alpha-peptide coding sequence of the enzyme beta-galactosidase. Insertion of a PCR product (or any DNA fragment for that matter) between cloning sites in the alpha-peptide region causes inactivation of beta-galactosidase, which otherwise is expressed and produces blue coloration seen clearly in the bacterial colonies. Therefore, successfully cloned colonies can be selected based on the lack of beta-galactosidase activity, which has been inactivated by the insert. This blue/white selection screen, in addition to antibiotic selection adds to the robustness of the pGEM-T vector systems. The selected white colonies can then be picked from the plate, cultured up to required quantities and used as reservoirs of the plasmid carrying the cloned fragment of interest.

Promega provides a very comprehensive booklet that accompanies the kit. It gives good background to the product and the technology, as well as instructions on how to culture cloned cells, even when the bacterial cells are not provided. The protocol also contains useful details about the plasmid design and restriction sites. The experimental procedure itself involves a DNA ligation step in which the PCR product, pGEM-T vector, ligase buffer and T4 DNA ligase enzyme are incubated, ideally overnight at 4°C. Competent cells are then transformed with the pGEM-T vectors using standard transformation protocols and colonies carrying the clones are identified by blue/white screening using X-GAL/IPTG LB agar plates. In our hands, the plates are quite stable in the fridge or cold room for a month or two. Overall, an easy to use kit that consistently delivers.