Abstract for pGEM®-T and pGEM®-T Easy Vector Systems
Part# TM042
Printed in USA. Revised 6/09.
Instructions for Use of Products A1360,
A1380,
A3600 &
A3610.
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.
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