pGEM-T Vector System I

Efficient PCR Cloning with Blue/White Selection

The pGEM®-T Vector Systems are convenient systems to clone PCR products generated by certain thermostable polymerases. These polymerases often add a single deoxyadenosine, in a template-independent fashion, to the 3´-ends of the amplified fragments. The pGEM®-T Vector is ready to use in ligation reactions, prepared by cutting the pGEM®-5Zf(+) Vector with EcoRV and adding a 3´ terminal thymidine to both ends. These single 3´-T overhangs at the insertion site greatly improve the efficiency of ligation of a PCR product into the plasmid by preventing recircularization of the vector and providing a compatible overhang for ligation of PCR products with A overhangs.

T7 and SP6 RNA polymerase promoters flank a multiple cloning region within the ?-peptide coding region for ?-galactosidase. Insertional inactivation of the ?-peptide allows recombinant clones to be directly identified by Blue/White Screening on indicator plates. The vector allows preparation of single-stranded DNA due to its f1 Origin of Replication.

The multiple cloning site is flanked by recognition sites for the restriction enzyme BstZI, allowing release of the insert by a single-enzyme digestion. Alternatively, a double digestion may be used to release the insert from the vector.

The pGEM®-T Vector System II contains JM109 Competent Cells in addition to all of the pGEM®-T Vector System I components.

1. Mezei, L.M. and Storts, D.R. (1994) In: PCR Technology: Current Innovations, Griffin, H.G. and Griffin, A.M., eds., CRC Press, Boca Raton, FL, 21.
2. Robles, J. and Doers, M. (1994) Promega Notes 45, 19–20.
3. Clark, J.M. (1988) Nucl. Acids Res. 16, 9677–86.
4. Newton, C.R. and Graham, A. (1994) In: PCR, BIOS Scientific Publishers, Ltd., Oxford, UK, 13.