Transfection

From Genome@Yale

Lipofectamine 2000 Manual

HT29-APC

1. Seed 6 well plate at 10% confluency in McCoy’s 5A +10% FBS +0.6mg/mL Hygromycin
2. Next day, when cells are ~30% confluent, wash cells with McCoy’s 5A +10% FBS and start transfection
3. Set up transfection mix in eppendorf or falcon tubes - following is for one well, repeat for other wells in the plate:
   1. Dilute 4ug DNA in 0.25mL OPTIMEM (per well); vortex lightly
   2. Dilute 15uL Lipofectamine 2000 in 0.25mL OPTIMEM, vortex lightly
   3. 5 minutes at RT
   4. Mix 250uL DNA with 250uL Lipofectamine (vortex lightly)
   5. Incubate @ RT 20 minutes
4. Aspirate media from cells
5. Add 500uL OPTIMEM to each well
6. Add 500uL DNA-Lipofectamine mixture to each well, swirl around to mix
7. 5 hour incubation
8. Add 1mL OPTIMEM + 20% FBS O/N (24hrs)
9. Add 20uL 10mM ZnCl2 to 2mL media (100uM ZnCl2 final)
10. 12-24 hours incubation
11. Wash with PBS
12. Add 200uL Lysis solution
13. Transfer to eppendorf tubes
14. Centrifuge at maximum speed for 2 minutes
15. Transfer to new eppendorf tubes and stored at -80°C until assays (reporter, western blot) are performed

HeLa

1. Seed 6 well plate at 50% confluency in DMEM +10% FBS
2. Next day, when cells are ~80% confluent, wash cells with OptiMEM and start transfection
3. Pre-warm 50ml of OptiMEM (stored in cold room at 4°C) ~10min.
4. Add 4ug of DNA to Eppendorf tube in the hood.
5. Add 250uL of OptiMEM to the Eppendorf tube to dilute the DNA and mix by tapping.
6. Add 12uL of Lipofectamine2000 and 250uL of OptiMEM to another Eppendorf Tube.
7. Combine #3 (DNA) and #4 (Lipofectamine2000).
8. Mix well by tapping or vortexing slowly.
9. Spin down quickly at low speed (~3000 rpm).
10. Incubate for 20min at room temperature.
11. Add ~500uL #8 to the well gently and drop-wise.
12. Add 1mL OPTIMEM + 20% FBS
13. Incubate #9 for 48 hrs in the 37°C incubator
14. For stables, split 1:10 into flask and wait another 24 hours before adding antibiotics for selection.

HEK293

1. Seed 6 well plate at 50% confluency in DMEM +10% FBS
2. Next day, when cells are ~80% confluent, wash cells with OptiMEM and start transfection
3. Pre-warm 50ml of OptiMEM (stored in cold room at 4°C) ~10min.
4. Add 4ug of DNA to Eppendorf tube in the hood.
5. Add 250uL of OptiMEM to the Eppendorf tube to dilute the DNA and mix by tapping.
6. Add 12uL of Lipofectamine2000 and 250uL of OptiMEM to another Eppendorf Tube.
7. Combine #3 (DNA) and #4 (Lipofectamine2000).
8. Mix well by tapping or vortexing slowly.
9. Incubate for 20 minutes at room temperature.
10. Add ~500uL #8 to the well gently and drop-wise.
11. Add 1mL OPTIMEM + 20% FBS
12. Incubate for 48 hrs in the 37°C incubator
13. For stables, split 1:10 into flask and wait another 24 hours before adding antibiotics for selection.