... Protocols Lentivirus Production Lentivirus Production You may also like.... Timeline Day 0: Seed 293T packaging cells Day 1 (pm): Transfect packaging cells Day 2 (am): 18 h post-transfection...are below passage 15 for viral production. Procedure Seed 293T packaging cells at 3.8×10 6 cells per plate...plasmid DNA purification should include an endotoxin removal step. For high quality plasmid DNA, the plasmids...the diluted DNA mixture. Add the diluted PEI dropwise while gently flicking the diluted DNA tube. Incubate...This protocol can be used to produce lentivirus from a lentiviral vector transfected into 293T cells using...for alternative packaging cell lines or transfection reagents. Once produced, lentivirus can be used for...

...measure the DNA concentration, use 1 μL) 2 μL 10x NEB T4 DNA ligase buffer 1 μL NEB T4 DNA ligase add ...Producing Lentiviral Particles E.1 Recommended materials E.2 Protocol for producing lentiviral particles...#B7001S NEB buffer 2 NEB #B7002S T4 DNA ligase NEB #M0202S T4 DNA ligase buffer NEB #B0202S DH5 alpha ...will increase the chance of damaging the DNA. Purify the DNA using a Qiaquick gel extraction kit. Elute...kit to obtain DNA. 3. Conduct a restriction digest with EcoRI and NcoI: 1 μg miniprep DNA 2 μL 10x NEB ...transfect plasmid DNA directly into the target cells. The shRNA will be expressed, but the DNA is unlikely ...optimal puromycin concentration F.3 Protocol for lentiviral infection and selection G. Safety H. References...