cool to room temperature. To each 90 mL aliquot of the base
medium, add 10 mL of OADC enrichment (BD Difco),
0.1 mL of 10% Pan solution (see Note 1), and 250 μL of 20%
v/v Tween 80 (final concentration of 0.05% v/v).
4. Sauton’s medium base with pan: Dissolve 0.5 g KH 2 PO 4 , 0.5 g
MgSO 4 , 4 g L-asparagine monohydrate, 2 g citric acid, and
0.05 g ferric ammonium citrate to 900 mL deionized water.
Add 60 mL glycerol. Adjust pH to 7.0 with 1 M NaOH.
Autoclave and store at room temperature. Add 1 mL of 10%
Pan solution per liter of the medium before use (see Note 1). If
culturing planktonic cells, add 2.5 mL of sterile 20% Tween
80 per liter of medium before use.
5. High-zinc Sauton’s medium with pan: Add 1 mL of sterile 1 M
ZnSO 4 ∙7H 2 O (see item 5 of Subheading 2.1) per liter of sterile
Sauton’s medium base with pan.
6. PBSTw: See item 8 of Subheading 2.1.
7. Centrifuge and rotor similar to Thermo Sorvall Legend XTR
with Thermo Scientific Swing-out rotor and corresponding
adapters and tubes.
2.3 Cell Lysis
1. 0.1% diethyl pyrocarbonate (DEPC)-treated water stock: Add
1 mL of DEPC to 1 L of doubled-distilled water. Autoclave
and store at room temperature.
2. 1 M HEPES-KOH pH 7.5: Dissolve 23.83 g of HEPES in
80 mL of DEPC-treated water. Adjust the pH to 7.5 with
1 M KOH. Adjust the volume to 100 mL with DEPC-treated
water. Filter-sterilize the solution and store at 4
C.
3. 1 M ammonium chloride (NH 4 Cl): Dissolve 5.3 g of NH 4 Cl in
90 mL of DEPC-treated water. Adjust the volume to 100 mL
with DEPC-treated water. Filter-sterilize the solution and store
at 4
C.
4. 1 M magnesium chloride (MgCl 2 ): Dissolve 9.5 g of MgCl 2 in
90 mL of DEPC-treated water. Adjust the volume to 100 mL
with DEPC-treated water. Filter-sterilize the solution and store
at 4
C.
5. 12.5 M β-mercaptoethanol (BME): Commercially sourced
molecular biology grade.
6. 500 mL low-salt HMA buffer: 20 mM HEPES-KOH, 10 mM
MgCl 2 , 30 mM NH 4 Cl, 5 mM BME. To 470 mL of DEPCtreated water, add 10 mL of 1 M HEPES-KOH pH 7.5, 15 mL
of 1 M NH 4 Cl, 5 mL of 1 M MgCl 2, and 5 mM BME. Filtersterilize the solution and store at 4
C.
7. Liquid nitrogen.
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Yunlong Li et al.
medium, add 10 mL of OADC enrichment (BD Difco),
0.1 mL of 10% Pan solution (see Note 1), and 250 μL of 20%
v/v Tween 80 (final concentration of 0.05% v/v).
4. Sauton’s medium base with pan: Dissolve 0.5 g KH 2 PO 4 , 0.5 g
MgSO 4 , 4 g L-asparagine monohydrate, 2 g citric acid, and
0.05 g ferric ammonium citrate to 900 mL deionized water.
Add 60 mL glycerol. Adjust pH to 7.0 with 1 M NaOH.
Autoclave and store at room temperature. Add 1 mL of 10%
Pan solution per liter of the medium before use (see Note 1). If
culturing planktonic cells, add 2.5 mL of sterile 20% Tween
80 per liter of medium before use.
5. High-zinc Sauton’s medium with pan: Add 1 mL of sterile 1 M
ZnSO 4 ∙7H 2 O (see item 5 of Subheading 2.1) per liter of sterile
Sauton’s medium base with pan.
6. PBSTw: See item 8 of Subheading 2.1.
7. Centrifuge and rotor similar to Thermo Sorvall Legend XTR
with Thermo Scientific Swing-out rotor and corresponding
adapters and tubes.
2.3 Cell Lysis
1. 0.1% diethyl pyrocarbonate (DEPC)-treated water stock: Add
1 mL of DEPC to 1 L of doubled-distilled water. Autoclave
and store at room temperature.
2. 1 M HEPES-KOH pH 7.5: Dissolve 23.83 g of HEPES in
80 mL of DEPC-treated water. Adjust the pH to 7.5 with
1 M KOH. Adjust the volume to 100 mL with DEPC-treated
water. Filter-sterilize the solution and store at 4
C.
3. 1 M ammonium chloride (NH 4 Cl): Dissolve 5.3 g of NH 4 Cl in
90 mL of DEPC-treated water. Adjust the volume to 100 mL
with DEPC-treated water. Filter-sterilize the solution and store
at 4
C.
4. 1 M magnesium chloride (MgCl 2 ): Dissolve 9.5 g of MgCl 2 in
90 mL of DEPC-treated water. Adjust the volume to 100 mL
with DEPC-treated water. Filter-sterilize the solution and store
at 4
C.
5. 12.5 M β-mercaptoethanol (BME): Commercially sourced
molecular biology grade.
6. 500 mL low-salt HMA buffer: 20 mM HEPES-KOH, 10 mM
MgCl 2 , 30 mM NH 4 Cl, 5 mM BME. To 470 mL of DEPCtreated water, add 10 mL of 1 M HEPES-KOH pH 7.5, 15 mL
of 1 M NH 4 Cl, 5 mL of 1 M MgCl 2, and 5 mM BME. Filtersterilize the solution and store at 4
C.
7. Liquid nitrogen.
154
Yunlong Li et al.