Protocol for Transducing Mouse Embryonic Fibroblasts (MEF) using MISSION® ExpressMag™
Super Magnetic Kit®
Protocol courtesy of Sigma MISSION RNAi Team
- Healthy rapidly dividing mouse embryonic fibroblasts (MEF) cells
- ExpressMag Super Magnetic Kit (SHM01)
- 0.25% Trypsin EDTA solution (T3924)
- Puromycin Solution (P9620)
- DMEM (D5671)
- MEF cell media – DMEM (D5671) supplemented with L-glutamine, 10% FBS (F0926) and nonessential amino acids (M7145)
- MEF cell media supplemented with puromycin at 3 mg/ml (although puromycin concentration may need to be optimized for your particular cell line)
- Tissue culture incubator — 37 °C, 5% CO2, 100% relative humidity
- Tissue culture laminar flow hood—only open optimization plate in tissue culture hood
- MISSION lentivirus (SHC002V in example)
0.1 Seed 15k MEF per well into 96-well plate. Utilize a final volume of 200 µl of media per well. (Note: for best data interpretation always include an untransduced well that is treated with puromycin. There should be few to no surviving cells in this well at assay time.) For gene silencing experiments, total time in puromycin will have to be empirically determined, depending upon the half life of the protein and mRNA.
0.2 Incubate overnight.
1.1 Calculate volume of virus (Vv) in μl per well for an MOI of 8.
1.2 Utilizing the following formula calculate the volume of DMEM (Dv) needed per well to bind virus to beads then add DMEM to empty tube. Dv=50-Vv-1.5
1.3 Add 1.5 μl of ExpressMag beads (SHM03) per well to the DMEM.
1.4 Add virus to the mixture and gently mix by pipetting the full volume.
1.5 Incubate for 15 minutes at room temperature – do not disturb during incubation.
1.6 Remove 50 μl from each well to be transduced.
1.7 Add 50 µl of mixture from step 1.5 to each well to be transduced.
1.8 Incubate at room temperature for 15 minutes on super magnetic plate (SHM04)
2.1 (Morning) Replace media with MEF cell media.
2.2 (Evening) Add puromycin to a final concentration of 3 μg/ml
3.1 (Morning) Replace media with MEF cell media supplemented with puromycin.
4.1 (Morning) Replace media with MEF cell media supplemented with puromycin.
4.2 (Evening) Assay cells
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