Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Isotope Labeling 257
pad of folded cheesecloth and cover with a double layer of aluminum foil. Autoclave.
After autoclaving, this can be stored a day or two at room temperature or
for several days at cold room temperature. The other ingredients should be made
up, sterilized separately, and added just prior to inoculation.
2. TY Medium, (1 L):
a. Bacto Tryptone (16 g)
b. Bacto Yeast Extract (8 g)
c. NaCl (5 g)
d. Appropriate antibiotics
e. pH = 7.5
Dissolve all ingredients except antibiotics in 1 L ddH 2O and sterilize by autoclaving.
Add antibiotics before inoculation.
3. NZ Medium, (1 L):
a. NZ amine (10 g)
b. Bacto Tryptone (5 g)
c. NaCl (5 g)
d. MgSO 4 (2.5 g) (see Note 6)
e. Appropriate antibiotics
f. pH = 7.5
Dissolve all ingredients except antibiotics and MgSO 4 in 1 L ddH 2O and sterilize
by autoclaving. Add antibiotics and MgSO 4 before inoculation.
4. IPTG (Isopropyl β-D-Thiogalactopyranoside): Dissolve 120 mg into 8 mL of
water and filter-sterilizes. This is enough for a 1-L prep.
5. Antibiotics:
a. 5% ampicillin: Weigh out and dissolve in water. Filter sterilizes into a sterile
container. Store in freezer. This stock can be used for up to ten days but breaks
down over time or heating up above 55°C. Use 1 µL per 1 mL of medium.
b. 2.5% chloramphenicol: Weigh out with a sterile spatula and dissolve into 99%
ethanol in a sterile test tube or other sterile container. This solution cannot be
sterilized by either autoclave or filter since it breaks down with heat and ethanol
breaks down the filter membrane in filter-sterilizers. This stock can be
kept frozen up to 6 mo. Use 1 µL per 1 mL of medium.
c. Other antibiotics: Follow specific protocols for specific antibiotics.
6. Vitamins and Trace Elements Mixture (see Note 8): Weigh out 10 mg per 1 mL
each of biotin, choline chloride, folic acid, niacinamide, D-pantothenic acid, and
pyridoxal chloride and 1 mg per 100 mL of riboflavin in H 2O. All above can be
mixed and made as a single batch, then filter-sterilized, or made individually and
filters sterilized as desired. Store frozen and in the dark. Use 10 mL of above
mixture per 1 L of medium
25% of nicotinc acid in H 2O, filter-sterilizes separately. Use 2.5 mL per 1 L of
medium.
10% of thiamine in H 2O, filer sterilizes separately. Use 0.5 mL per 1 L of medium.