Summary: 9. Protocol for Preparation of ES Cells for Blastocyst Injection
UCSD Transgenic Core Facility (Updated 5/13/02) Contact Ella Kothari at 822-3754.
Injection Media Recipe: DMEM with hepes and 5% fetal bovine serum
-Invetrogen (Gibco) DMEM high glucose & 25 mM HEPES, 500ml (smallest size) Cat #12430-
-1 ml frozen aliquots of (inactivated) ES compatible FBS
Pipet 19 mls injection DMEM to sterile conical and add 1 ml FBS mix. Syringe filter through .2um filter
(discard first ml or so). Expel remaing to new tube, cap and set on ice.
1.) Thaw and plate MEF's to a gelatinized 6 well plate
2.) Thaw and plate one vial of ES cells onto 1 or 2 wells of the above 6 well plate.
Wednesday Feed those cells.
At 9 am (about 1.5 hours before you need the cells for injection), prep them:
1. Aspirate the medium.
2. Rinse three times with 2 ml PBS (without CaCl2 and MgCl2).
3. Add 5 drops of 0.05% trypsin-EDTA with a 5 ml pipet.
4. Incubate 5-7 minutes at 37 0
C in incubator.
5. Neutralize with 1 ml ES media, pipette the cells 30 times with a P1000 at 500ul to get single