Autoinduction Stock Solutions (for Protocol go here):
ZY
- 10 g N-Z-amine AS (or any tryptic digest of casein, e.g. tryptone)
- 5 g yeast extract
- Make to 1 litre with dH20
20x NPS
|
Component |
100 ml |
1 liter |
mol/liter |
|
dd H2O |
90 ml |
900 ml |
- |
|
(NH4)2SO4 |
6.6 g |
66 g |
0.5 M |
|
KH2PO4 |
13.6 g |
136 g |
1 M |
|
Na2HPO4 |
14.2 g |
142 g |
1 M |
- Add in sequence in beaker; stir until all dissolved.
- pH of 20-fold dilution in water should be ~6.75.
50x5052: (5052 = 0.5 % glycerol, 0.05% glucose, 0.2% α-lactose)
|
Component |
100 ml |
1 liter |
|
Glycerol (weigh in beaker) |
25 g |
250 g |
|
H2O |
73 ml |
730 ml |
|
Glucose |
2.5 g |
25 g |
|
a-lactose |
10 g |
100 g |
- Add in sequence in beaker, stir until all dissolved
- Lactose is slow to dissolve — may take two hours or more of stirring. Brief heating in a microwave can speed up dissolution of the lactose.
1 M MgSO4
- 24.65 g MgSO4•7H2O
- Water to make 100 ml
40% glucose (w/v)
|
Component |
100 ml |
300 ml |
|
Glucose |
40 g |
120 g |
|
H2O |
74 ml |
222 ml |
- Add glucose to stirring water in beaker; DO NOT ATTEMPT TO ADD WATER TO GLUCOSE!
- Stir until all dissolved — may take 45 minutes or more of stirring.
80% glycerol (v/v) (= 100% w/v)
- 100 g glycerol (weigh in beaker)
- 20 ml water
20% α-lactose (w/v)
|
Component |
100 ml |
600 ml |
|
a-lactose |
20 g |
120 g |
|
H2O |
87.5 ml |
525 ml |
- add lactose to stirring water in beaker
- stir until all dissolved -- may take 2 hours or more
1000x trace metals mixture (100 ml in ~50 mM HCl)
Prepare all metal stock solutions in ddH2O, except for FeCl3, which is dissolved in ~0.1M HCl, as noted in the table below. Combine the metal solutions as in the table below.
|
Component |
Vol |
MW |
1x conc |
|
H2O |
36 ml |
- |
- |
|
0.1 M FeCL3•6H2O (dissolved in ~0.1 M HCl = 100-fold dil of conc HCl) |
50 ml |
270.30 |
50 µM Fe |
|
1M CaCl2 |
2 ml |
110.99 |
20 µM Ca |
|
1M MnCl2•4H2O |
1 ml |
197.91 |
10 µM |
|
1 M ZnSO4•7H2O |
1 ml |
287.56 |
10 µM Zn |
|
0.2 M CoCl2•6H2O |
1 ml |
237.95 |
2 µM Co |
|
0.1 M CuCl2•2H2O |
2 ml |
170.486 |
2 µM Cu |
|
0.2 M NiCl2•6H2O |
1 ml |
237.72 |
2 µM Ni |
|
0.1 M Na2MoO4•2H2O |
2 ml |
241.98 |
2 µM Mo |
|
0.1 M Na2SeO3•5H2O |
2 ml |
263.03 |
2 µM Se |
|
0.1 M H3BO3 |
2 ml |
61.83 |
2 µM H3BO3 |
Autoclave the stock solutions of the individual metals, except 0.1 M FeCl3 in 1/100 volume conc HCl. A brief precipitate may appeared upon addition of Na2SeO3, however it will redissolve rapidly. Store at room temperature
When making growth media, add the metals mix before NPS. If NPS is already present when 1000x metals mix is added, a precipitate forms which disperses but retains a light turbidity. If the metals are diluted to near their final concentration before NPS is added, the medium remains clear. The metals also precipitate and disperse or redissolve when added to ZY, a precipitate caused by yeast extract. Although apparently not a problem, the precipitate could be avoided by diluting the metals in the water before dissolving the yeast extract in making ZY.
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