Trypsin-EDTA (1x), stored frozen

ERDC TN-DOER-C30

February 2003

b) When thawed, dry outside of the container with paper towel and wipe with

paper towel moistened with 70 percent alcohol.

c) Aseptically transfer 50-mL aliquots of FBS into sterile centrifuge tubes.

d) Cap tubes and store in -20 C freezer until further use.

4) Trypsin-EDTA (1x), stored frozen.

a) Remove 10x trypsin-EDTA stock (100 ml) from -20 C freezer and place in

37 C water bath.

b) When thawed, dry outside of container with paper towel and wipe with paper

towel moistened with 70-percent alcohol.

c) Aseptically transfer the 10x trypsin-EDTA into a sterile 1-L bottle containing

900 mL of sterile-filtered water and mix contents well.

d) Transfer 12-mL aliquots of 1x trypsin-EDTA into 15-mL sterile centrifuge

tubes.

e) Cap tubes and store in -20 C freezer.

5) Hygromycin B, 100-mg/mL or 500x solution, stored refrigerated.

a) Transfer 1.5-mL aliquots in sterile cryovials.

b) Store in the refrigerator.

6) Zeocin, 100-mg/mL or 500x solution, stored frozen.

a) This antibiotic comes in 8 sterile aliquots of 1.25 mL.

b) Store in the -20oC freezer.

NOTE: The Hygromycin B and Zeocin concentration needed to maintain

selection pressure on these recombinant cells is 0.2 mg/mL. In a T-75 flask

containing 15 mL of medium, add 30 L (or 0.2 percent of medium volume) each

of the 100-mg/mL Hygromycin B and 100-mg/mL Zeocin stock. Both

Hygromycin B and Zeocin are added to the medium in every cell passage.

7) Trypan Blue, 0.05 percent, stored refrigerated, nonsterile.

a) Transfer 6.3 mL of 0.4 percent Trypan Blue solution (stored at room

temperature) into 43.7 mL of PBS solution in a beaker and mix well.

b) Pipet 1.800 mL of the 0.05 percent Trypan Blue solution with a micropipettor

into each of the 5-mL culture tubes.

CAUTION: Trypan Blue is a suspected mutagen. Wear gloves at all times

when handling this reagent.

c) Cover the neck of each tube with a piece of parafilm.

d) Place tubes in a rack and store in refrigerator.

8) Freezing Cell Medium, made fresh each time.