Transfection protocol for 6-well dish

1. Seed cells at appropriate density (5x10⁴/well for 3T3, MPAC, BHK; 5x10⁵/well for aTC1.6, bTC3) one to two days before transfection.
2. Combine total DNA at 1ug /well with 600ul/well serum-free medium in an eppendorf tube
3. Add 1.5ul/well Transfast (Promega) to the DNA/medium mixture. Vortex for 10 seconds
4. Incubate at RT for 10 to 15 min
5. During the above incubation, aspirate medium from cells and add PBS; remove PBS just before adding DNA in step 6.
6. Add 600ul DNA mixture from step 3 to the appropriate well.
7. Incubate at 37 ^oC incubator for 1 hr.
8.  Add 4ml regular growth medium to each well
9. after 24 hours, change to fresh medium
10.  After 48 hrs, harvest cells for functional assay