One shot top10 competent e coli cells

The full-length hCB1R gene (1.4 kbp) was amplified from pRC/CMV CB1 construct as template, using Pfu DNA polymerase (Stratagene) under the following thermocycling conditions: 95 °C for 1 min, followed by 30 cycles of 95 °C for 30 s and 68 °C for 2 min, followed by an extension time of 5 min at 68 °C in an Eppendorf Mastercycler (Westbury, NY). NheI and BamHI restriction sites were incorporated into forward 5′-CGCTAGCATG-AAGTCGATCCTAGATGGCCT-3′ and reverse 5′-TATGGATCC-TCACAGAGCCTCGGCAGACGTG-3′ primers, respectively. The PCR product was purified using a MinElute PCR kit (Qiagen) and was digested using BamHI and NheI restriction enzymes. The same restriction enzymes were used for digestion of pcDNA5/FRT expression vector (Invitrogen). The vector and PCR fragment were purified using a MinElute kit and ligated at room temperature for 1 h. The ligated products were then transformed into One Shot Top10 competent E. coli cells following the vendor’s protocol (Invitrogen). Plasmid preparations were cultured in Luria broth containing 0.1 mg/mL ampicillin. Recombinant plasmid DNA was isolated using a pure link kit (Invitrogen), and DNA insertion was confirmed by sequencing (University of Connecticut Biotechnology Center, Storrs, CT).