Abstract for pGEM®-T and pGEM®-T Easy Vector Systems
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 color screening on indicator plates. The multiple cloning
region of the two vectors includes restriction sites conveniently arranged for
use with the Erase-a-Base® System (Cat.# E5750) for
generating nested sets of deletions.
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. |
