EZ Rich Defined Medium

Updated 5/5/03. Now this medium is available commercially.

Updated 6/14/02. This is our protocol for preparing Neidhardt Supplemented MOPS Defined Medium. It is called "EZ" because modifications were made to the details of the protocol from Neidhardt's lab to streamline it ( i.e. Neidhardt's lab recipe called for fresh cysteine, but we mix the cysteine with the other amino acids in the frozen supplement solution) and to correct some minor errors.

Reference: F. C. Neidhardt, P. L. Bloch, and D. F. Smith. 1974. Culture medium for enterobacteria. J Bacteriol 119(3): 736-747 [PubMed Central].

EZ Rich Defined Medium (EZ RDM)

10X MOPS Mixture 100ml

0.132 M K2HPO4

10 ml

10X ACGU

100 ml

5X Supplement EZ

200 ml

sterile H2O

580 ml

TOTAL

990 ml

Before use add 100X carbon source (as specified): 10 ml

EZ Rich Defined Medium Plates (EZ RDM Plates)

10X MOPS Mixture 100 ml

0.132 M K2HPO4

10 ml

10X ACGU

100 ml

5X Supplement EZ

200 ml

sterile H2O

80 ml

TOTAL

490 ml

In a separate 2 L flask, mix 13 g Bacto Agar and 500 ml of water. Autoclave 20 minutes on liquid cycle. Add the mixture above to the flask within about 15 minutes of removing it from the autoclave. Add 10 mL of 100X carbon source (usually specified, but if not, use 20% glucose) and antibiotics (if specified). Pour immediately.

10X MOPS mixture

1. In a 1 L beaker with a stir bar, add the following to ~300 ml milliQ H2O:

Component FW grams

MOPS

209.3

83.72

Tricine

179.2

7.17

2. Add 10 M KOH to a final pH of 7.4 (10 to 20 ml)
3. Bring total volume to 440 ml
4. Make fresh FeSO4 solution and add it to the MOPS/Tricine solution:

Component FW grams H2O vol (ml) stock conc (M)
FeSO4•7H2O

278

0.028

10 0.01

5. Add the following solutions to the MOPS/Tricine/FeSO4 solution (see below how to make each of these):

Mix in the order shown!

Component Volume

1.9 M NH4Cl

50 ml

0.276 M K2SO4

10 ml

0.02 M CaCl2•2H2O

0.25 ml

2.5 M MgCl2

2.1 ml

5 M NaCl

100 ml

Micronutrient stock

0.2 ml

Autoclaved milliQ H2O

387 ml

TOTAL

1000 ml

6. Filter sterilize with 1 L capacity 0.2 micron filter
7. Aliquot into sterile 100 or 200 ml plastic bottles and freeze at -20 degrees.

Stocks used in 10X MOPS mixture

Make each separately, mixing the amount indicated into the specified volume. Store at room temp.

Component FW stock conc (M) grams vol (ml) note

NH4Cl

53.49

1.9

50.82

500

 

K2SO4

174.3

0.276

4.8

100

 

CaCl2•2H2O

147

0.02

0.294

100

 

MgCl2

203.3

2.5

50.75

100

 

NaCl

58.44

5

292.2

1000

 

Micronutrient stock (50 ml)

Mix everything together in 40 ml autoclaved MQ-H2O, bring up total volume to 50 ml. Store at room temp.

Component Formula FW Grams for 50 ml

ammonium molybdate

(NH4)6Mo7O24•4H2O

1235.9

0.009

boric acid

H3BO3

61.83

0.062

cobalt chloride

CoCl2

237.9

0.018

cupric sulfate

CuSO4

249.7

0.006

manganese chloride

MnCl2

197.9

0.040

zinc sulfate

ZnSO4

287.5

0.007

Potasium phosphate K2HPO4 Solution

NOTE!! Make sure you use dibasic K2HPO4, and do NOT use monobasic KH2PO4 !!!!! Can be stored at room temperature after autoclaving.

Component FW stock conc. (M) grams Vol (ml) note
K2HPO4

173.2

0.132

23.0 1000 Autoclave

10X ACUG (1000 ml)

1. In a 1 L beaker with stir bar, add 1.5 ml 10 M KOH to 1 L milliQ H2O to make 0.015 M KOH
2. Dissolve the following together:

Component FW Grams for 1000 ml Note

adenine

135.13

0.270

Dissolve together in 0.015 M KOH

cytosine

111.1

0.222

Filter sterilize

uracil

112.09

0.224

Store at -20° C

guanine

151.13

0.302

 

3. Filter sterilize with 1 L capacity 0.2 micron filter
4. Aliquot into sterile 100 or 200 ml plastic bottles and freeze at -20° C .

5X Supplement EZ (1000 ml)

Mix together the following solutions (see below for how to make each). Filter sterilize with 1 L capacity 0.2 micron filter. Aliquot into sterile 100 or 200 ml plastic bottles and freeze at -20° C.

Stock solution

Volume (ml)

alanine (free)

5

arginine (HCl)

65

asparagine (free)

40

aspartic acid (K salt)

5

cysteine (HCl H2O)

50

glutamic acid (K salt)

5

glutamine (free)

25

glycine (free)

5

histidine (HCl H2O)

5

isoleucine (free)

10

leucine (free)

80

lysine

5

methionine (free)

5

phenylalanine (free)

40

proline (free)

5

serine (free)

125

threonine (free)

5

tryptophane (free)

10

tyrosine (free)

100

valine (free)

10

VA Vitamin Solution 50
H2O 350

TOTAL

1000

Amino acid stocks used in 5X Supplment

Make each separately, mixing the amount indicated into the specified volume. Store at -20° C.

Name

FW

Weight (g)

Vol (ml)

Notes

alanine (free)

89.09

1.78

25

 

arginine (HCl)

210.70

8.44

100

 

asparagine (free)

132.10

0.66

100

 

aspartic acid (K salt)

171.20

1.71

25

 
cysteine (HCl H2O) 175.60 0.09 50 will oxidize to cystine, so 5X supplement and RDM should be frozen

glutamic acid (K salt)

185.30

2.78

25

 

glutamine (free)

146.20

1.8

100

 

glycine (free)

75.07

1.50

25

 

histidine (HCl H2O)

209.60

1.05

25

 

isoleucine (free)

131.20

0.65

25

 

leucine (free)

131.20

0.66

100

 

lysine

182.70

1.83

25

 

methionine (free)

149.20

0.75

25

 

phenylalanine (free)

165.20

0.83

100

 

proline (free)

115.10

1.15

25

 

serine (free)

105.10

4.20

100

 

threonine (free)

119.10

1.19

25

 

tryptophane (free)

204.20

0.26

25

 

tyrosine (free)

181.20

0.045

25

dissolve tyrosine in 0.01 M KOH

valine (free)

117.20

0.88

25

 

100X VA Vitamin Solution (500 ml)

Mix together the following solutions (see below for how to make each): Store at -20° C.

Stock solution

volume

0.02 M thiamine HCl

25

0.02 M calcium pantothenate

25

0.02 M p-aminobenzoic acid

25

0.02 M p-hydroxybenzoic acid

25

0.02 M 2,3-dihydroxybenzoic acid

25

milliQ H2O

375

TOTAL

500

Stock solutions used in 100X VA

Make each separately, mixing the amount indicated into the specified volume

Name

FW

grams

volume (ml)

 

thiamine HCl

337.3

0.169

25

 

calcium pantothenate

476.5

0.238

25

 

p-aminobenzoic acid

137.13

0.069

25

dissolve in 0.02 M KOH

p-hydroxybenzoic acid

138.13

0.069

25

dissolve in 0.02 M KOH

2,3-dihydroxybenzoic acid

154.12

0.077

25

dissolve in 0.02 M KOH


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