...Producing Lentiviral Particles E.1 Recommended materials E.2 Protocol for producing lentiviral particles...protocol for using the pLKO.1 cloning vector, a backbone used by the RNAi consortium for targeting human...optimal puromycin concentration F.3 Protocol for lentiviral infection and selection G. Safety H. References...RNAi Consortium The pLKO.1 cloning vector is the backbone upon which The RNAi Consortium has built a library...of pLKO.1 pLKO.1 is a replication-incompetent lentiviral vector chosen by the TRC for expression of shRNAs...direct transfection, or can be converted into lentiviral particles for subsequent infection of a target...analog). Due to the long terminal repeats found in lentiviral plasmids, Addgene currently recommends using ...

...enzyme for digest that will cut the shared plasmid backbone a single time and visualize that digestion on ...gel ( see restriction digest protocol here ). Lentiviral plasmids can recombine between their LTRs resulting...these sequences are not efficiently packaged into lentiviral particles. See below for options if the recombinant...

...Cloning protocols for using the pLKO.1 vector, a backbone used by the RNAi consortium for targeting human...plasmid DNA to mammalian cells Lentivirus Production Produce lentivirus with a polyethyenimine (PEI) transfection...preparation Lentivirus Titration by ddPCR Use ddPCR to measure the physical titer of your lentivirus preparation...transfection protocol Fluorescence Titering Assay for Lentivirus Quantify virus using fluorescence measurements...measurements Colony Formation Titering Assay for Lentivirus Quantify virus by counting antibiotic resistant colonies...colonies Generating Stable Cell Lines with Lentivirus Genomically integrate your DNA sequence into a cell...