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Reagent Recipies

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CI Solution (24:1)	1% CTAB	0.5M EDTA	Fe-EDTA (A&A)	Iron Reducer Mineral Solution	Iron Solution	Microelement Stock
Micronutrient Solution	PCI Solution (25:24:1)	PES Stock	+Pi Stock	-Pi Stock	Proteinase K solution (20 mg/mL)	25% Ringer Solution
10% SDS	TAE	TBE	TE Buffer	TESC	Thiosulfate	Trace Elements for Heliobacteria
Trace Metal Solution A5+Co	Trace Metal Solution (IMR)	1M Tris	Vitamin Solution (IMR)	Vitamin Solution (PES30)

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CI Solution (24:1)

Isoamyl Alcohol	10 mL
Chloroform	240 mL
-Put on gloves, cover forearms, and relocate to fume hood -Add both components to a sterile 250 mL glass bottle -Mix well -Wrap container in foil to protect from light -Store at 4^oC

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1% CTAB

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0.5M EDTA

Na2EDTA- dihydrate	186.1 g
Milli-Q Water	~1 L
NaOH	~20 g
-Add EDTA into 800 mL Milli-Q water (NOTE: EDTA will not dissolve) -Using NaOH pellets, adjust pH to 8.0 (EDTA should now dissolve) -Bring volume to 1 L with Milli-Q -Autoclave for 30 minutes -Store at 4^oC

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Fe-EDTA (A&A)

Na2EDTA•2H2O	866.7 mg
FeSO4•7H2O or Fe2(SO4)3•nH2O	2.28 mg
KOH	866.7 mg
Milli-Q Water	~100 mL
-Dissolve KOH in 31 mL Milli-Q -Add Na2EDTA•2H2O -In separate container, dissolve FeSO4•7H2O or Fe2(SO4)3•nH2O in 60.67 mL Milli-Q -Mix both solutions together -Bubble filtered air through combined solutions until the color darkens (~2.5 hr)

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Iron Reducer Mineral Solution

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Iron Solution

FeNH4(SO4)2•??H2O	???
Na2EDTA	300 mg
Milli-Q Water	~500 mL
-Measure out 250 mL Milli-Q -Add components in order -Bring volume up to 500 mL -Autoclave for 25 minutes

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Microelement Stock

Fe-EDTA solution	80 mL
MnCl2•4H2O	180 mg
MoO3 (85% purity) or Na2Mo4•2H2O (99% purity)	18 mg 30.55 mg
ZnSO4•7H2O	22.0 mg
CuSO4•5H2O	7.9 mg
H3BO3 (Boric Acid)	286 mg
Vanadyl sufate hydrate	6.6 mg
CoC12•6H2O	4.0 mg
Milli-Q Water	~545 mL
-Measure out Milli-Q -Dissolve all components in order (Note: Do Not autoclave)

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Micronutrient Solution

H2SO4	500 µL
H3BO3	500 mg
MnSO4•H2O	2.28 g
ZnSO4•7H2O	500 mg
CuSO4•5H2O	25 mg
Na2MoO4•2H2O	25 mg
CoCl2•6H2O	45 mg
Na2SeO4	4 mg
Milli-Q Water	~1 L
-Measure out 500 mL of Milli-Q -Dissolve all components in order -Bring volume to 1 L with Milli-Q

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PCI Solution (25:24:1)

Isoamyl Alcohol	5 mL
Chloroform	120 mL
Phenol	125 mL
-Put on gloves, cover forearms, and relocate to fume hood -Combine all components in sterile 250 mL glass bottle -Mix well -Wrap container in foil to protect from light -Store at 4^oC

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PES Stock Solution

NaNO3	50 mg
Na2-Glycerolphosphate•4H2O	50 mg
Tris Buffer	500 mg
Iron Solution	25 mL
Trace Metal	15 mL
Milli-Q Water	100 mL
-Measure out Milli-Q -Add NaNO3 and Na2-Glycerolphosphate•4H2O -Combine trace metal and iron solutions in separate container -Autoclave both containers -Once cool, combine both mixtures -Store at 4^oC in 10 mL aliquots

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+Pi Stock

2HPO4•3H2O	28 g
K2HPO4, anhydrous	21.4 g
Milli-Q Water	500 mL
-Measure out Milli-Q -Add either of two chemicals -Autoclave both containers -Store at 4^oC

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-Pi Stock

MgSO4•7H2O	20 g
CaCl2•2H2O	6 g
NaCl	20 g
Microelement Stock	500 mL
Milli-Q Water	1.5 L
-Dilute first three ingredients separately into 500 mL of Milli-Q -Autoclave three solutions, and allow to cool -Combine four solutions in order, allowing each to dissolve -Store at 4^oC, shake well before use, do not autoclave

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Proteinase K Solution (20 mg/mL)

Proteinase K	20 mg
Milli-Q Water	1 mL
-Dissolve proteinase K into Milli-Q -Filter sterilize and dispense into 1.5 mL Eppendorf tube -Store at -20^oC

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25% Ringer Solution

NaCl	2.25 g
KCl	105 mg
CaCl2	45 mg
NaHCO3	50 mg
Citric Acid	34 mg
Milli-Q Water	~1 L
-Measure out approximately 300 mL Milli-Q -Add all components in order -Bring volume up to 1 L with Milli-Q -Autoclave for 45 minutes

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10% SDS

Sodium Dodecyl Sulfate (SDS)	25 g
Milli-Q Water	~250 mL
-Pour 175 mL Milli-Q into sterile 250 mL glass bottle -Put on mask (very important) and weigh out SDS -Thoroughly clean countertop and balance -Add SDS to the Milli-Q and heat to dissolve -Bring volume to 250 mL with Milli-Q -Adjust pH to 7.2 using sterile HCL (1% v/v) and/or NaOH (1% w/v) -Loosen cap and microwave for one to two minutes -Store at room temperature

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TAE

Trizma Base	4.844 g
EDTA	372.2 mg
Glacial Acetic Acid	1.15 mL
Milli-Q Water	~1 L
-Measure out approximately 300 mL Milli-Q -Add all components in order -Bring volume up to 1 L with Milli-Q -Adjust pH to 8.3 -Store at 4^oC

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TBE

Trizma Base	12.11 g
Boric Acid, anhydrous	5.565 g
EDTA	372 mg
Milli-Q Water	~1 L
-Measure out approximately 300 mL Milli-Q -Add all components in order -Bring volume up to 1 L with Milli-Q -Adjust pH to 8.3 -Store at 4^o

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TE Buffer

1M Tris	5 mL
0.5M EDTA	1 mL
Milli-Q Water	~500 mL
-Measure out 490 mL Milli-Q -Add components in order -Adjust pH to 7.4 using HCl -Bring volume to 500 mL -Autoclave for 30 minutes -Store at 4^oC

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TESC

NaCl	87.66 g
1M Tris	100 mL
0.5M EDTA	200 mL
Hexadecyltrimethylammonium Bromide (CTAB)	10 g
Milli-Q Water	~1 L
-Measure out approximately 500 mL Milli-Q -Add all components in order -Bring volume up to 1 L with Milli-Q -Adjust pH to ~8.0 -Autoclave for 30 minutes -Store at room temperature

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Thiosulfate

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Trace Elements for Heliobacteria

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Trace Metal Solution A₅+Co

H3BO3	2.86 g
MnCl2•4H2O	1.81 g
ZnSO4•7H2O	222 mg
Na2MoO4•2H2O	390 mg
CuSO4•5H20	79 mg
Co(NO3)2•6H2O	49 mg
Milli-Q Water	~1 L
-Measure out 500 mL Milli-Q -Add components in order -Bring volume to 1 L with Milli-Q

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Trace Metal Solution (IMR)

CuSO4•5H2O (9.80 g/L)	1 mL
ZnSO4•7H2O (22 g/L)	1 mL
CoCl2•6H2O (10 g/L)	1 mL
MnCl2•7H2O (244 g/L)	1 mL
Na2MoO4•7H2O (6.3 g/L)	1 mL
Fe2(SO4)3•nH2O (900 mg/L)	500 mL
EDTA•Na2 (6.7 g/L)	500 mL
-Combine last two ingredients -Add all other ingredients in order -Store at 4^oC

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1M Tris

Tris Base	121.1 g
Milli-Q Water	~1 L
HCl	~42 mL
-Dissolve Tris base into 800 mL Milli-Q water -Using HCl, adjust pH to 8.0 -Bring volume to 1 L using Milli-Q -Autoclave for 30 minutes (NOTE: re-make if solution turns yellow) -Store at 4^oC

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Vitamin Solution (IMR)

Thiamine•HCl (20 g/L)	1 mL
Biotin (50 mg/L)	2 mL
Vitamin B12 (1 g/L)	100 µL
Milli-Q Water	96.9 mL
-Combine all ingredients -Filter sterilize the solution -Store at 4^oC

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Vitamin Solution (PES30)

Biotin	10 mg
Thiamine	200 mg
Cyanocobalamin	10 mg
Folic Acid	10 mg
Myo-Inositol	10 mg
Calcium Pantothenate	100 mg
Milli-Q Water	~1 L
-Measure out 500 mL Milli-Q -Add components in order -Bring volume up to 1 L with Milli-Q -Filter sterilize before use

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