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 ERDC TN-DOER-C30
February 2003
The following reagents are prepared in bulk ahead of time and stored in aliquots in the refrigerator
or freezer. Contents and date of preparation should be properly labeled and initialed on these reagent
containers.
B. REAGENT PREPARATION
1) Minimum Essential Medium (MEM) - supplemented with L-glutamine and FBS;
stored refrigerated.
a) Remove L-glutamine (100x) and FBS from refrigerator or freezer and place
in 37 C water bath.
IMPORTANT: As a precaution to avoid cell contamination, the water bath
should be cleaned and the water changed at least once a month.
b) When warmed, dry outside of containers with paper towel and wipe with
paper towel moistened with 70 percent alcohol.
c) Under a sterile flow hood, remove 55 mL of MEM from a new bottle. This
unsupplemented medium can be saved for later use (such as in the making of
the cell freezing medium). Aseptically, transfer the following components
into the 500-mL bottle.
Component
Volume, mL
Percent
MEM
445
89
FBS
50
10
L-Glutamine (100x)
5
1
d) Refrigerate the fully supplemented medium when not in use.
IMPORTANT: L-glutamine should be replenished at 1 percent by volume
of the remaining medium in the bottle every 3 weeks.
2) Sterile-filtered, ultra-pure water, stored refrigerated.
a) Place a 500-mL beaker of ultra-pure water (not sterile) in the hood.
b) Attach the bottle-top (0.1- m VacuCap90) filter to a sterile, autoclaved
(500-mL) bottle.
c) Connect tubings on the filter to the aspirator and to the beaker containing
ultra-pure water.
d) Turn on aspirator.
e) When the bottle is filled, turn off the aspirator and disconnect the tubings.
f)
This filtered water can be stored in the refrigerator until further use.
3) Fetal Bovine Serum (FBS), stored frozen.
a) Remove FBS from freezer and place in 37 C water bath.
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