Geneamp pcr system 9700 machine

All the primers were designed using Primer Premier 5.0 (Premier Biosoft, CA; Table 1). Polymerase chain reaction (PCR) amplification of conserved nucleotide regions for each gene (cry1: Cry1F and Cry1R; cry2: Cry2F and Cry2R) were performed on GeneAmp PCR System 9700 machine (Applied Biosystems, Foster City, CA) using the following conditions: 94°C preincubation for 5 min; 94°C for 45 s, 60°C for 45 s, 72°C for 2 min, for 35 cycles; and 72°C final extension for 10 min. PCR products were inserted into the pEASY-T3 vector (TransGen Biotech, Beijing, China) and sequenced by Taihe Biotechnology Company (Beijing, China).
According to the procedures of rapid amplification of cDNA end technique, the full-length cDNAs of cry genes were obtained. Briefly, the 5′- and 3′-ends of cry genes receptors were amplified using the universal primer mix (BD Biosciences, San Jose, CA) with specific primers (Table 1). PCR thermal cycling conditions were 94°C for 5 min; 35 cycles of 94°C for 30 s, 65°C for 5′-RACE, 65°C for Cry1-3′-RACE, 68°C for Cry2-outer-3′-RACE and 60°C for Cry2-inner-3′-RACE 30 s, and 72°C for 1 min; and 72°C for 10 min. All PCR products were cloned into pEASY-T3 vector and sequenced as detailed already.