DMEM Protocol
Materials
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1000 mL beaker
150 mL beaker
1000 mL nanopure water
DMEM powder (High Glucose - Sigma D5648)
3.7 g sodium bicarbonate (49.3 mL) per liter of DMEM being prepared (Sigma
S5761)
1 N HCl
1 N NaOH
pH meter
bottle top filter with porosity of 0.22 µm
2 500 mL bottles (autoclaved)
Procedure
1. Wipe down hood with alcohol.
2. Put sterilized bottles and bottle top filter under hood.
3. Measure out 90% of the final volume required (900 mL) of nanopure water into the
1000 mL beaker. Measure out the rest of the volume (100 mL) into 150 mL beaker.
Water temperature should be 15° - 20° C.
4. Add DMEM powder slowly to the 1000 mL beaker while stirring gently. Stir until
dissolved. DO NOT HEAT.
5. Rinse original DMEM package with a small amount of nanopure water from the 150
mL beaker. Add this to the solution and any remaining nanopure water in step 4.
6. Add 3.7 g of sodium bicarbonate to solution in step 5. Stir until dissolved.
7. While stirring, adjust pH of solution using 1 N HCl and 1 N NaOH until it at the
desired pH (7.4).
8. Sterilize the prepared solution (DMEM) by sterile filtering using a bottle top filter
into 2 500 mL sterile bottles in the hood.
9. Label bottles with the following:
• DMEM
• Sterile
• Date
• Initials
10. Refrigerate.
Verified May 25, 2004 J.O. Modified 2-25-13