...Transfection grade DNAs: Lentiviral Packaging plasmid(s), Envelope plasmid, and Transfer plasmid encoding your insert...1.3 pmol Envelope Plasmid ( pMD2.G ) 0.72 pmol Transfer Plasmid* 1.64 pmol OptiPro SFM to total volume...transfection plasmids into OptiPro SFM: Reagent Amount per 10 cm dish* Packaging Plasmid ( psPAX2 ) 1.3... Timeline Day 0: Seed 293T packaging cells Day 1 (pm): Transfect packaging cells Day 2 (am): 18 h post-transfection...volume 500 μL *Plasmid concentrations and ratios should be optimized for each transfer plasmid. Pro-Tip Endotoxins...therefore, plasmid DNA purification should include an endotoxin removal step. For high quality plasmid DNA, ... cm dish). Using transfer plasmid pHAGE TRE dCas9-KRAB (total μg of plasmid DNA 27.8 μg), this would be...

...μg pLKO.1 shRNA plasmid 750 ng psPAX2 packaging plasmid 250 ng pMD2.G envelope plasmid to 20 μl serum-... shRNA. psPAX2 Packaging plasmid for producing viral particles. pMD2.G Envelope plasmid for producing ...to vary the ratio of shRNA plasmid, packaging plasmid, and envelope plasmid to obtain the ratio that gives....1 can also be used with packaging plasmid pCMV-dR8.2 dvpr and envelope plasmid pCMV-VSVG from Robert Weinberg...Producing Lentiviral Particles E.1 Recommended materials E.2 Protocol for producing lentiviral particles...Due to the long terminal repeats found in lentiviral plasmids, Addgene currently recommends using a strain...puromycin concentration F.3 Protocol for lentiviral infection and selection G. Safety H. References H.1 Published...