Endofree plasmid purification giga kit

Glycerol stocks of the plasmid constructs were thawed and streaked on to LB ampicillin agar plates. After overnight incubation at 37℃, a single colony was transferred with a sterile inoculating loop into 5 mL of LB ampicillin broth and incubated at 37℃ for 6-8 hours. The starter culture was subsequently inoculated into 50 mL of fresh LB ampicillin broth and grown at 37℃ with vigorous shaking (500 rpm) overnight, which was further inoculated into 2.5 L of fresh LB ampicillin broth and incubated under similar conditions. After 16 hours of growth, the bacteria were pelleted by centrifugation at 5,000 rpm at 4℃. The plasmid identity was once again verified by restriction enzyme analysis of a plasmid miniprep made from 1.5 mL of the overnight culture. Large-scale purification of each plasmid was then performed from the bacterial pellets using a commercial kit (EndoFree Plasmid Purification Giga Kit, Qiagen), as per manufacturer instructions. The quality and concentration of the plasmid preparations were evaluated by spectrophotometry and agarose electrophoresis, and the preparations were stored in aliquots at -20℃ till further use.

The bicistronic eukaryotic expression vector pIRES was a gracious gift from Dr Praveen K Gupta, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India. The plasmid pBacPak-GRC9 containing the full-length rabies virus glycoprotein of a street virus isolate (human) was earlier produced in the Department of Neurovirology, National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore, India, as part of another project. EndoFree Plasmid Purification Giga Kit (QIAGEN, Düsseldorf, Germany) was used for the large-scale, endotoxin-free purification of the plasmid construct, as per manufacturer instructions.