SET UP NEW 24-WELL PLATES
- 1. Get two new 24-well plates
- 2. Label the lid so that the top 12 wells are “no puro” and the bottom 12 are “plus puro”
- 3. Each plate will be used for BD004 or BD006. Label accordingly
- 4. Aliquot 1 mL U-2 OS complete (no puro) into each appro. Well (both plates)
- 5. Aliquot 1 mL U-2 OS complete plus puro (0.5 ug/ml) into each appro. Well (both plates)
- 6. Place in incubator is you need to pause your work after this step
- 1. Pick your favorite 12 wells from each set. You will discard the remainder
- 2. Aspirate off old medium. Change pipettes when switching to new plate.
- 3. Wash cells with 0.5 mL 1xPBS
- 4. Aspirate off PBS
- 5. Detach cells with 200 uL Trypsin-EDTA (note: first pipette about 5 mL Trypsin-EDTA into a 15 mL conical, then use the P-1000 micropipettor to add trypsin to wells….never use a micropipettor in stock bottles for TC work)
- 6. Incubate at RT for ~3 min.
RE-PLATE THE CELLS
- 7. Pipette 10 mL of 1 mL U-2 OS complete (no puro) into a 15 mL conical (to avoid using the micropipettor in the medium stock bottle).
- 8. Resuspend cells with 1 mL U-2 OS complete (no puro) using a P-1000 micropipettor.
- 9. Set the micropipettor to 100 uL and add 100 uL resuspended cells to a no-puro well, then add another 100 uL resuspended cells to a plus-puro well.
- 10. Discard (by aspiration) any leftover cells, aliquotted medium, etc. into vacuum trap.