Endo free plasmid dna mini kit

Total RNA from GF-1 cells were extracted cells using Trizol (Invitrogen, Japan) and transcribed into cDNAs using SuperScript II reverse transcriptase kit (Invitrogen, USA) according to the manufacturer's instructions. PCR reactions were performed using primer sets given in Table 1, detected by 1.0% agarose gel electrophoresis, purified and ligated into a pMD18-T simple vector (Takara, Dalian, China), as described earlier. The full-length cDNA encoding GF-CypA was PCR amplified using the modified primers with restriction enzymes (BamH1 and EcoRI; bold and underlined) 5′-GAATTCAGATTGTCATGGAGCTGAGA-3′ and 5′-GGATCCGCAGTCTGTGATCACGATCTT-3′, were purified to clone into eukaryotic expression plasmid pcDNA3.1 (Invitrogen). Purified PCR fragments and the pcDNA3.1 plasmid were digested with the corresponding restriction enzymes then ligated together. The recombinant plasmid was transformed into Escherichia coli BL21 (TransGen, Beijing, China). Plasmid pcDNA3.1-CypA was purified using Endo-free Plasmid DNA Mini Kit (Omega bio-tek, Norcross, Georgia), quantified and stored in −20 °C, until further analysis.