Protocol:CDM
Solution I
NaCL ……………………23.38 g
K2SO4……………….…… 4.00 g
NH4CL…………………....0.88 g
Na2EDTA……………….0.012 g
Dissolve Na2EDTA in about 50 ml of 0.1 N NaOH
Then add the rest of the reagents and continue stirring until reagents go into solution
q.s. to 100 ml in ddH2O, filter sterilize, and store at RT
Solution II
K2HPO4…………………..17.42 g
KH2PO4…………….…….13.61 g
q.s. to 100 ml in ddH2O, filter sterilize, and store at RT
Solution III
L-alanine………………….0.80 g
L-arginine…………..…….1.20 g
L-asparagine……………….0.2 g
L-aspartic acid……………4.00 g
q.s. to 100 ml ddH2O and stir until reagents go into solution (warm on hot plate if
necessary)
L-cysteine HCL………….0.44 g
L-cystine HCL…….……..0.28 g
q.s. to 100 ml ddH2O and stir until reagents go into solution (warm on hot plate if
necessary)
L-glutamic acid………….10.40 g
L-glutamine……………… 0.40 g
L-glycine………………….0.20 g
L-histidine HCL…………..0.20 g
L-isoleucine………………0.24 g
L-leucine………………….0.72 g
L-lysine……………………0.40g
q.s. to 100 ml ddH2O and stir until reagents go into solution (warm on hot plate if
necessary)
L-methionine……………..0.12 g
L-phenylalanine….……….0.20 g
L-proline………………….0.40 g
L-serine……………..…….0.40 g
L-Threonine………………0.40 g
q.s. to 100 ml ddH2O and stir until reagents go into solution (warm on hot plate if
necessary)
L-tryptophan………………0.64 g
L-valine……………………0.48 g
q.s. to 100 ml ddH2O and stir until reagents go into solution (warm on hot plate if
necessary)
Glutathione…………………0.36 g
(4dessicator)
L-tyrosine…………………..0.56 g
(4Cdessicator)
q.s. to 100 ml ddH2O and stir until reagents go into solution (warm on hot plate if
necessary)

Let all fractions cool to RT and combine

q.s. to 1000 ml in ddH2O, filter sterilize, and store at 4C protected from light

note:use hydrochlorides if available as they are more soluble
Solution IV
Thiamine HCL………………0.020 g
Thiamine pyrophosphate….…0.005g
(also called cocarboxylase: -20Cdessicator)
Pantothenic acid……………..0.019 g
(4Cdessicator)
d-biotin………………………0.030 g

q.s. to 100 ml in 50% ethanol, filter sterilize, and store at -20C
Solution V
Dextrose (20%)…….…………20.0 g

q.s. to 100 ml in ddH2O, filter sterilize, and store at 4C
Solution VI
Hypoxanthine…………………0.50 g
Uracil………………………….0.50 g

q.s. to 100 ml in 0.1 N NaOH, filter sterilize, and store at -20C
1M CaCl2
CaCl2 …..………….………27.75 g

q.s. to 250 ml with ddH2O in an Fe- treated or polypropylene bottle, autoclave and
store at RT
1M MgCl2
MgCl2 6 H2O …..…………50.83 g

q.s. to 250 ml with ddH2O in an Fe- treated or polypropylene bottle, autoclave and
store at RT
500 mM sodium bicarbonate
NaHCO3………………..21 g

q.s. to 500 ml with ddH2O in an Fe- treated or polypropylene bottle, filter sterilize
into an Fe- treated or polypropylene bottle and store at RT.
10mM ferric nitrate
Fe(NO3)3…………0.404 g

q.s. to 100 ml with ddH2O, filter sterilize and store at RT.
4X CDM Stock Preparation
4X CDM Stock
Solution 1………………...…….50 ml
Solution II……….………………4 ml
Solution III……………………250 ml
Solution IV………………..……20 ml
Solution V……………..……….50 ml
Solution VI……………………..20 ml
Add 20 g HEPES, stir until dissolved and pH to 7.5
q.s. to 500 ml with ddH2O
4X CDM stock solution must have the iron extracted using Chelex
prepare Chelex by weighing out 30 g, wash two times with 2 liters of ddH2O using a
Buchner funnel, remove most of the water
add the washed Chelex to the 500 ml of CDM and stir for 90 minutes
filter sterilize into an Fe- treated or polypropylene bottle and store at 4C
CDM-0
4X CDM
ddH2O
500mM NaHCO3
25 ml
71 ml
4 ml
100 ml

prepare in an Fe- treated bottle

add 100 ul of 1M MgCl2

add 25 ul of 1M CaCL2

note: MgCl2 and CaCL2 must be added in this order or CDM-0 will turn cloudy
and will need to be remade.
CDM-10 (10uM final iron conc)
4X CDM
ddH2O
500mM NaHCO3
25 ml
71 ml
4 ml
100 ml

prepare in an Fe- treated bottle

add 100ul of 1M MgCl2

add 25ul of 1M CaCL2

note: MgCl2 and CaCL2 must be added in this order or CDM-10 will turn cloudy
and will need to be remade.

Add 100ul of 10 mM Fe(NO3)3
Fe- treated glassware

Place glassware in a 3N nitric acid bath for at least 24hrs

Rinse with ddH2O at least 10 times

Let glassware dry and autoclave
Fe- treated ddH2O

Add ddH2O to an Fe- treated bottle and autoclave

Use this water to make up CDM-0, CDM-10, 1M MgCl2, CaCL2, and 500mM
NaHCO3