shRNA Lentivirus Production using HEK293T cells andFuGENEBiohazard ConcernsLentivirus is a modified HIV virus and although unable to replicate in a host, it must be handled with caution. When working with these viruses, work only in BL2+ designated hoods or viral vector rooms. All handling, storage and disposal of biohazard waste must be in accordance with Institute rules and regulations, OSHA, EPA and MWA.NotesAll rules of the virus room must be followed. Be sure you receive the proper training on policies and procedures of the virus room before use.Materials1.)293T packaging cells (ATCC # CRL-11268)2.)Plasmid DNA for:a.)shRNA-pLKO.1 plasmidb.)pCMV-dR8.91 (Delta 8.9) plasmid containing gag, pol and rev genesc.)VSV-G expressing envelope plasmidd.)pLKO.ps control plasmid3.)FuGENE 6 (Fisher # NC9666789 )4.)100mm Corning CellBIND tissue culture plates (Fisher # 07-202-516)5.)DMEM media (ATCC #30-2002)6.)OptiMem Reduced Serum Media (Invitrogen # 31985-070)7.)FBS (ATCC #30-2020)8.)Pen/Strep (ATCC #30-2300)9.)15mL Centrifuge Tubes (Fisher # 05-538-53F)10.) 50mL Centrifuge Tubes (Fisher # 07-203-510)
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