Cell Signaling PCR Cloning pGEM®-T Vector Systems Product pGEM®-T Vector System I pGEM®-T Vector System II Size Cat.# Price (Fr) 20 reactions A3600 289.00 20 reactions A3610 527.00 For Research Use Only. Not for Use in Diagnostic Procedures. Description: The pGEM®-T Vector Systems are convenient systems for the cloning of PCR products. The pGEM®-T Vector is 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 generated by certain thermostable polymerases. These polymerases often add a single deoxyadenosine, in a templateindependent fashion, to the 3´-ends of amplified fragments. 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. Features: • Rapid Ligation: The 2X Rapid Ligation Buffer provided allows reactions to be completed in 1 hour at room temperature. • Blue/White Screening: 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 color screening on indicator plates. • f1 Origin of Replication: Allows the preparation of single-stranded DNA. Storage Conditions: Store competent cells at –70°C; store all other components at –20°C. Protocol pGEM®-T and pGEM®-T Easy Vector Systems Technical Manual XmnI 1994 ScaI 1875 f1 ori Ampr pGEM® Vector (3000bp) -T NaeI 2692 T7 lacZ T T ApaI AatII SphI BstZI NcoI SacII ori SpeI NotI BstZI PstI SalI NdeI SacI BstXI NsiI SP6 14 20 26 31 37 46 103 112 55 62 62 73 75 82 94 126 1 start ori XmnI 2009 ScaI 1890 Part# TM042 Ampr f1 ori pGEM®-T Easy Vector (3015bp) lacZ NaeI 2707 T7 T T ApaI AatII SphI BstZI NcoI BstZI NotI SacII EcoRI SpeI EcoRI NotI BstZI PstI SalI NdeI SacI BstXI NsiI SP6 1. Add PCR product to T-Vector. 2. Add 2X Rapid Ligation Buffer and T4 DNA Ligase. 2,000 1,800 1,600 1,400 1,200 1,000 800 600 400 200 0 14 20 26 31 37 43 43 49 52 1 start pGEM®-T Easy Vector Systems Product pGEM®-T Easy Vector System I pGEM®-T Easy Vector System II Size Cat.# Price (Fr) 20 reactions A1360 318.00 20 reactions A1380 556.00 For Research Use Only. Not for Use in Diagnostic Procedures. Description: The pGEM®-T Easy Vector Systems are convenient systems for the cloning of PCR products. They offer all of the advantages of the pGEM®-T Vector Systems with the added convenience of recognition sites for EcoRI and NotI flanking the insertion site. Thus several options for removal of the desired insert DNA with a single restriction digestion are provided. The pGEM®-T Easy Vector System II contains JM109 Competent Cells in addition to all of the pGEM®-T Easy Vector System I components. Features: • Flexible: The multiple cloning site is flanked by restriction enzyme sites for BstZI, NotI and EcoRI, allowing three options for removal of the insert with a single digest. • Rapid Ligation: The 2X Rapid Ligation Buffer provided allows reactions to be completed in 1 hour at room temperature. • Blue/White Screening: 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 color screening on indicator plates. • f1 Origin of Replication: Allows the preparation of single-stranded DNA. Storage Conditions: Store competent cells at –70°C; store all other components at –20°C. Protocol pGEM®-T and pGEM®-T Easy Vector Systems Technical Manual Part# TM042 12 109 118 127 141 64 70 77 77 88 90 97 Recombinants 90% Recombinant colonies Nonrecombinant colonies Recombinants 67% Unpurified Purified 4. Transform Competent Cells. 3. Incubate 1 hour. The rapid ligation reaction reduces ligation time to just 60 minutes. For complete and up-to-date product information visit: www.promega.com/catalog 221 Purification of PCR products enhances cloning success. A 500bp PCR product was purified with the Wizard® SV Gel and PCR Clean-Up System and cloned into the pGEM®-T Easy Vector. Both the percent recombinants and total number of colonies increase with a pure PCR product. White bars represent recombinant colonies. Blue bars represent nonrecombinant colonies. PCR    1985MD 0356VA04_3A Total Colonies  1473VA05_6A 4382MA11_3A Pagina 224

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