Lentiviral Transfection


Posted by xwang
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Protocol

This protocol is for transfection to produce pseudotyped lentivirus using FuGene 6 transfection reagent in a 10cm dish.

Materials: 
  • 1.2ml microcentrifuge tubes
  • 10 cm plate of 293 cells (Falcon)
  • 30 ul fugene (Roche)
  • 600 ul Serum Free MEM or DMEM (Gibco)
  • 4 ug of packaging vector master mix- equal parts of pVSV-G, pMDL, pRSV
  • 4 ug of lentiviral plasmid
Methods: 
  1. In 1.5 ml tubes mix 600ul SFMEM with 30ul FuGene. (Note: do not allow the fugene to come into contact with the tube in its undiluted form.  Refer to manufacturers instructions)
  2. In cap mix lentiviral plasmid with packaging vector master mix
  3. Close lid and mix
  4. Incubate 20 minutes at room temperature
  5. Pour into 293 plate
  6. Allow viral production to continue for 48-96 hours before harvest (Note: Virus producing cells have a distinct rounded phenotype)
  7. Harvest and or concentrate (see Lentiviral harvest and concentration protocol)