pGEM®-T and pGEM®-T Easy Vector Systems Protocol
Literature # TM042
The pGEM®-T and pGEM®-T Easy Vector Systems are convenient systems for the cloning of PCR products. The vectors are prepared by cutting the pGEM®-5Zf(+) and pGEM®-T Easy Vectors, respectively, with EcoR V 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 plasmids by preventing recircularization of the vector and providing a compatible overhang for 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 high copy number pGEM®-T and pGEM®-T Easy Vectors contain T7 and SP6 RNA polymerase promoters flanking a multiple cloning region within the alpha-peptide coding region of the enzyme beta-galactosidase. Insertional inactivation of the alpha-peptide allows recombinant clones to be directly identified by blue/white screening on indicator plates.
The pGEM®-T and pGEM®-T Easy Vector Systems include a 2X Rapid Ligation Buffer for ligation of PCR products. Reactions using this buffer may be incubated for 1 hour at room temperature. The incubation period may be extended to increase the number of colonies after transformation.
Summary of Changes
The 12/18 version of this Technical Manual was revised to remove references to discontinued products in the notes on sequencing primers in Section 5.B. and Section 5.D.
Printed in USA. Revised 12/18.