Transfection protocol for 6-well dish

Seed cells at appropriate density (5x104/well for 3T3, MPAC, BHK; 5x105/well for aTC1.6, bTC3) one to two days before transfection.

Combine total DNA at 1ug /well with 600ul/well serum-free medium in an eppendorf tube.

Add 1.5ul/well Transfast (Promega) to the DNA/medium mixture. Vortex for 10 seconds.

Incubate at RT for 10 to 15 min.

During the above incubation, aspirate medium from cells and add PBS; remove PBS just before adding DNA in step 6.

Add 600ul DNA mixture from step 3 to the appropriate well.

Incubate at 37 oC incubator for 1 hr.

Add 4ml regular growth medium to each well.

after 24 hours, change to fresh medium.

After 48 hrs, harvest cells for functional assay.