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
|
|