One shot stbl3 e coli cells

FUG-W-pHRed DNA transfer plasmid was received as a gift from Dr. Tantama (Purdue University). A glycerol stock of E. coli transformed with the Perceval HR DNA transfer plasmid, FUG-W-Perceval HR, was purchased from Addgene (Cambridge, MA). Bacterial agar stabs carrying the accessory plasmids required for packaging and enveloping the virions, psPAX2 and pMD2.G, were also purchased from Addgene. We prepared a pHRed bacterial glycerol stock by transforming One Shot Stbl3 E. coli cells (Thermo Fisher, Waltham, MA) with FUG-W-pHRed DNA, following the manufacturer’s instructions. All glycerol stocks were then used to inoculate LB Broth (Sigma, St. Louis, MO) cultures grown with 100 μg/ml of ampicillin (Sigma) for about 15 h at 37 °C with a shaking incubator set at 225 rpm. DNA plasmids were extracted from these cultures and purified using the Endotoxin-free Maxiprep kit (Sigma). Their quantity and purity were measured with a Nanodrop spectrophotometer (Thermo Fisher). Plasmid identities were confirmed by 0.8% agarose gel electrophoresis using the E-Gel-iBase system (Thermo Fisher) following standard overnight restriction enzyme digestion by EcoRI (10 units/μg DNA; New England Biolabs, Ipswich, MA).