Protocol
Brief Description
This protocol is for transfection to produce pseudotyped lentivirus using FuGene 6 transfection reagent in a 10cm dish.
Materials
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
Methods
- 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)
- In cap mix lentiviral plasmid with packaging vector master mix
- Close lid and mix
- Incubate 20 minutes at room temperature
- Pour into 293 plate
- Allow viral production to continue for 48-96 hours before harvest (Note: Virus producing cells have a distinct rounded phenotype)
- Harvest and or concentrate (see Lentiviral harvest and concentration protocol)
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