Isolating Transfected U2OS BD004/BD006 Colonies
- With the BD006 1:10 plate and BD004 1:10 plate:
- Split 1:10 into another 10 cm plate and
- Split 1:10 into a second 10 cm plate (in effect, a 1:100 split).
- These plates will be used to determine if we can separate positive from negative cells by FACS.
- With both 1:20 plates:
- Removed old media and added standard media with 0.5 ug/mL puromycin to see if this is enough to kill off negative cells.
- With 1:40 plates:
- Made a grid on a piece of tape and stuck it to the bottom of the 10 cm plate.
- Used fluorescent microscope and grid to write down approximate location of groups of fluorescent cells.
- Acquired 17 locations for both plates; set up TC hood.
- Took out one plate at a time, washed with 5 mL of PBS, then added 5 mL of trypsin and let sit in the hood at RT for 5 min without moving the plate at all.
- During the 5 minutes, placed 900 uL of standard U2OS medium in 17 wells in a 24-well plate.
- After the 5 minutes were over, took 50 uL of trypsin and cells from the location written down without moving the plate, then took another 50 uL from the same location.
- Repeated this for the other 16 wells and the other plate.