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  1. Take the medium out.
  2. Wash with PBS once (10 ml/10cm plate; 5 ml/6cm plate). Washing is just putting solution in and out of the pate. Don’t shake the plate. Use suction to asprate the medium from the plate.
  3. Add trypsin (2ml/10cm plate; 1ml/6cm plate). Make sure that the bottom of the plate is evenly covered with trypsin. Tilt the plate with cells on one side and add the trypsin. Right after trypsin is added, tilt plate on the opposite side to distribute the trypsin evenly.
  4. Place the plate in the incubator for 15 minutes. After 15 minutes agitate the plate by moving it from side to side (left to right then forward to back) while plate is still on the incubator shelf. Never tilt the plate to one side after you take it out of the incubator. You do not want the cells to come off the as a sheet, you want single cells instead. Observe the plate under the microscope. You will probably see some clumping, which is fine.
  5. Take fresh ES cell medium into a pipette (8ml/10cm dish or 4ml/6cm dish). Do not disperse medium into the plate yet. Tilt the plate too the side, pick up the cells in the trypsin solution with the corresponding pipette containing a corresponding amount of medium. Once all the cells and the medium are in the pipette, gently disperse the content of the pipette back into the dish. GENTLY pipet it up and down 2-3 more times. Observe celles under the microscope. Most of the cells should be single cells at this point.
  6. Put the plate back in the incubator for 15-20 minutes. During this time the feeder cells will attach to the bottom of the plate while the ES cells will stay in the suspention.
  7. Pick up the cells from the plate with corresponding pipette size and place them into the round bottom 14 ml centrifuge tube.
  8. Centrifuge for 5 minutes at 270g
  9. Take the medium out, leaving about ~100 microliters in the centrifuge tube.
  10. Resuspend the pellet in about 1ml of fresh ES cell medium.

ES cell medium without Lif and b-mercaptoethanol:
DMEM high glucose medium
15% Fetal Bovine serum (heat deactivated for 30 minutes at 56 degrees)
pen/strep solution 6 ml
non-essential amino acids 6 ml
sodium piruvate 6 ml
L-glutamine 6 ml