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Autoinduction Media Components

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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 MgSO47H2O
  • 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 FeCL36H2O

(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 MnCl24H2O

1 ml

197.91

10 µM

1 M ZnSO47H2O

1 ml

287.56

10 µM Zn

0.2 M CoCl26H2O

1 ml

237.95

2 µM Co

0.1 M CuCl22H2O

2 ml

170.486

2 µM Cu

0.2 M NiCl26H2O

1 ml

237.72

2 µM Ni

0.1 M Na2MoO42H2O

2 ml

241.98

2 µM Mo

0.1 M Na2SeO35H2O

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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