1. Cell culture reagents: DMEM, Fetal bovine serum, penicillin-streptomycin
2. Rretrovirus solution prepared with pBabe-puro plasmid
3. Trypsin-EDTA solution
4. Polybrene.
5. Puromycin
6. 10cm tissue culture dishes
7. Pippets
8. Cloning cylinders
9. Laminar flow hood

Before infection, make a kill curve of puromycin on the cells to be infected by growing cells in different concentration of puromycin and set a selecting concentration at which the cells can just be completely killed.

Day 0:

1. Seed cells to be infected the day before infection (should be ~30% confluent when they are infected. For RK3E cells, use 5x10⁵/10cm dish).

Day 1:

2. Remove the media from RK3E cells.
3. Add the volume of retrovirus solution needed and add 10% FBS DMEM to 9 ml for 10cm plate onto RK3E cells.
4. Add 18ul of sterile polybrene (Sigma H9268, stock conc. 5 mg/ml in PBS = 500X ) to final conc. 10ug/ml and swirl to mix.
5. Place back at 37 o C incubator for 15 hrs.

Day 2:

6. Chang media with 10 mls of fresh media.

Day 3 and later

7. At least 36 hr after infection, replace media with the media containing selective drug.
   Replace media containing selective drug until resistant colonies appear and the control cells in the plate without infection are completely killed.
8. Colonies can be picked up with cloning cylinders and cells can be grown in fresh plate/flask.