1.      Measure out 90% of final volume.                       9L ddH₂O

Use TC only! Bucket (It's in the glassware cabinet)

2.      Add powdered DMEM to H₂O while stirring gently.

Rinse original package w/ddH₂O to remove trace amounts.

DMEM powder is in the 4° walk in.

Do NOT heat .

put cover on to prevent dust from contaminating.

Stir until dissolved.

3.      Add 3.7 g of Sodium Bicarbonate for each liter being prepared.

10 L à 37 g NaHCO₃

4.      Adjust pH to 0.1 - 0.3 units below desired pH.

·        pH will rise 0.1-0.3 units following filter sterilization.

·        Our desired pH is 7-7.2  à We pH to 6.9, hoping for about 7.2 after filtering.

Use 1N HCl or 1N NaOH as necessary (Andres forgot?)

5.      Add ddH₂O to final volume (10 L)

6.      Set up tissue culture hood for filter sterilization.

                                                                          i.      Turn lights, power, blowers on.

                                                                        ii.      Sterilize the surface with 70% Ethanol.

                                                                      iii.      Put bottles in hood, loosen all caps.

1.      500 ml sterile TC bottles (orange caps) ONLY.

7.      Set up Millipore Sterivac (cat # SKGPM10RJ) - Refer to manufacturers instructions (see accompanying literature)

Notes: - Set up with vacuum trap in-line between sterivac and house vacuum line.

-Do not tilt sterivac - this can cause spillage

8.      Bring DMEM solutions into tissue culture room on cart,

·        Do not put in tissue culture hood,

·        Place vacuum line into DMEM, connect other end to Sterivac,

9.      Filter ~500ml into each tissue culture bottle

·        If any contamination is suspected- THROW OUT!

10.  Secure caps tightly and store in 4° walk in.

11.   Test several random bottles for contamination by placing in 37° incubator overnight.

12.  If contamination is present on any of the bottles test ALL remaining bottles.