PCR Cloning with Blue/White Selection and Easy Insert Excision

The pGEM®-T Easy 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 Easy pre-linearized Vector contains 3´-T overhangs at the insertion site to provide a compatible overhang for PCR products. The provided 2X Rapid Ligation Buffer allows reactions to be completed in 1 hour at room temperature. The incubation period may be extended to increase the number of colonies after transformation.  

T7 and SP6 RNA polymerase promoters flank a multiple cloning region within the α-peptide coding region for β-galactosidase. Insertional inactivation of the α-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 pGEM®-T Easy Vector Systems offer all of the advantages of the pGEM®-T Vector Systems with the added convenience of recognition sites for BstZI, EcoRI and NotI flanking the insertion site. Thus, several options exist to remove the desired insert DNA with a single restriction digestion.

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