Lentiviral Transfection Protocol for a 10cm dish


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Protocol
Materials: 

10 cm plate of 293T cells (Falcon)

18 ul Mirus LT1 tranfection reagent

500 ul Serum Free MEM or DMEM (Gibco)

3 ug of packaging vector master mix- equal parts of pVSV-G, pMDL, pRSV-Rev

3 ug of lentiviral plasmid

Methods: 

*The day before, plate 3.0 x 10^6 293T cells

1.     In 1.5 ml tubes mix 500ul SFMEM with 18 ul Mirus and incubate for 5 minutes at room temperature

2.     In cap mix lentiviral plasmid with packaging vector master mix

3.     Close lid and mix

4.     Incubate 20 minutes at room temperature

5.     Pipette into 293T plate

6.     Allow viral production to continue for 72 hours before harvest (Note: Virus producing cells have a distinct rounded phenotype)

7.     Harvest and or concentrate (see Lentiviral harvest and concentration protocol)