Solution and Cell/Culture/Growth Media Preparations

Chelation Medium Preparation

To prepare 1L of Chelation Medium the following components are required.

Component	Amount	Concentration
NaCl (mw: 58.44 g/mol)	40.9 g	0.7 M
MgCl2·6H2O (mw: 203.3 g/mol)	6.1 g	0.03 M
MgSO4·7H2O (mw: 246.47 g/mol)	7.39 g	0.03 M
KCl (mw: 74.55 g/mol)	1.49 g	0.02 M
EGTA (mw: 380.35 g/mol)	7.6 g	0.02 M

Chelation Medium Preparation Steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 40.9 g of NaCl to the solution.
3. Add 6.1 g of MgCl2·6H2O to the solution.
4. Add 7.39 g of MgSO4·7H2O to the solution.
5. Add 1.49 g of KCl to the solution.
6. Add 7.6 g of EGTA to the solution.
7. Add distilled water until volume is 1 L.
8. Adjust pH to 5.5 using HCl. Autoclave or filter through a 0.2-µm membrane. Store at 4°C.

Digestion Medium Preparation 

To prepare 1L of Digestion Medium the following components are required.

Component	Amount	Concentration
NaCl (mw: 58.44 g/mol)	23.4 g	0.4 M
MgCl2·6H2O (mw: 203.3 g/mol)	26.4 g	0.13 M
MgSO4·7H2O (mw: 246.47 g/mol)	5.4 g	0.022 M
KCl (mw: 74.55 g/mol)	11.9 g	0.16 M
CaCl2 (mw: 110.98 g/mol)	300 mg	0.002 M
2-(N-morpholino)ethanesulfonic acid (mw: 195.24 g/mol)	1.95 g	0.01 M

Digestion Medium Preparation Steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 23.4 g of NaCl to the solution.
3. Add 26.4 g of MgCl2·6H2O to the solution.
4. Add 5.4 g of MgSO4·7H2O to the solution.
5. Add 11.9 g of KCl to the solution.
6. Add 300 mg of CaCl2 to the solution.
7. Add 1.95 g of 2-(N-morpholino)ethanesulfonic acid to the solution.
8. Add distilled water until volume is 1 L.
9. Adjust the pH to 6.5. Filter through a 0.2-µm filter and store at 4°C. Just before use, add 1% (w/v) cellulose (Sigma) and 3 U/mL alginate lyase (Boyen et al. 1990).

Embryonic Medium Preparation

Embryonic medium is used for culturing embryos. To prepare 1L of Embryonic Medium the following components are required.

Component	Amount	Concentration
NaCl (mw: 58.44 g/mol)	870 mg	0.015 M
KCl (mw: 74.55 g/mol)	40 mg	0.0005 M
CaCl2•2H2O (mw: 147.01 g/mol)	150 mg	0.001 M
MgSO4•7H2O (mw: 246.47 g/mol)	250 mg	0.001 M
KH2PO4 (mw: 136.09 g/mol)	20 mg	0.0002 M
Na2HPO4•12H2O (mw: 358.14 g/mol)	20 mg	0.0001 M
NaHCO3 (mw: 84.01 g/mol)	60 mg	0.0007 M

Embryonic Medium Preparation Steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 870 mg of NaCl to the solution.
3. Add 40 mg of KCl to the solution.
4. Add 150 mg of CaCl2•2H2O to the solution.
5. Add 250 mg of MgSO4•7H2O to the solution.
6. Add 20 mg of KH2PO4 to the solution.
7. Add 20 mg of Na2HPO4•12H2O to the solution.
8. Add 60 mg of NaHCO3 to the solution.
9. Add distilled water until volume is 1 L.
10. Adjust the pH to 7.0-7.5.

IPTG Solution Preparation

IPTG (isopropyl-beta-D-thiogalactopyranoside) is a synthetic lactose analog. To prepare 1L of IPTG Solution the following components are required.

Component	Amount	Concentration
IPTG (mw: 238.3 g/mol)	200 g	0.8393 M

IPTG Solution Preparation Steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 200 g of IPTG to the solution.
3. Add distilled water until volume is 1 L.
4. Sterilize by passing it through a 0.22-µm disposable filter. Dispense the solution into 1-ml aliquots and store them at -20°C.

M9 Minimal Salts Preparation

To prepare 1L of M9 Minimal Salts the following components are required.

Component	Amount	Concentration
Na2HPO4•7H2O (mw: 268.07 g/mol)	64 g	0.2387 M
KH2PO4 (mw: 136.09 g/mol)	15 g	0.1102 M
NaCl (mw: 58.44 g/mol)	2.5 g	0.0428 M
NH4Cl (mw: 53.49 g/mol)	5 g	0.0935 M

M9 Minimal Salts Preparation Steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 64 g of Na2HPO4•7H2O to the solution.
3. Add 15 g of KH2PO4 to the solution.
4. Add 2.5 g of NaCl to the solution.
5. Add 5 g of NH4Cl to the solution.
6. Add distilled water until volume is 1 L.
7. Divide the salt solution into 200-ml aliquots and sterilize by autoclaving for 15 minutes at 15 psi (1.05 kg/cm 2) on liquid cycle.

Modified Barth’s Saline (MBS) Preparation

Modified Barth’s Saline (MBS) is commonly used as culture media for Xenopus oocytes. To prepare 1L of Modified Barth’s Saline (MBS)s the following components are required;

Component	Amount	Concentration
HEPES (mw: 238.3 g/mol)	11.92 g	0.05 M
NaCl (mw: 58.44 g/mol)	51.43 g	0.88 M
KCl (mw: 74.55 g/mol)	750 mg	0.0101 M
NaHCO3 (mw: 84.01 g/mol)	2.1 g	0.025 M
MgSO4 · 7H2O (mw: 246.47 g/mol)	2.46 g	0.01 M

Modified Barth’s Saline (MBS) Preparation Steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 11.92 g of HEPES to the solution.
3. Add 51.43 g of NaCl to the solution.
4. Add 750 mg of KCl to the solution.
5. Add 2.1 g of NaHCO3 to the solution.
6. Add 2.46 g of MgSO4 · 7H2O to the solution.
7. Adjust the pH of 10x MBS salt solution to 7.8 with 10 M NaOH, and sterilize by autoclaving. Store indefinitely at room temperature.
8. Add distilled water until volume is 1 L.
9. To prepare 1x MBS, combine 100 mL of 10x MBS salt solution and 7 mL of 0.1 M CaCl2 with 893 mL of autoclaved deionized H2O. Add antibiotics. Store for up to 1-2 mo at 16°C-18°C (the same temperature used for oocyte incubation). Check before use.

Yeast Two Hybrid (Y2H) Media, Amino Acid Dropout Mixes Preparation

Amino acid dropout mixtures contain amino acid supplements necessary for the growth of yeast. This amino acid dropout mixture is suitable for use with yeast-two hybrid systems. To prepare 1L of Yeast Two Hybrid (Y2H) Media, Amino Acid Dropout Mixes the following components are required.

Component	Amount	Concentration
Adenine (mw: 135.13 g/mol)	10 mg	0.0001 M
L-Arginine (mw: 174.2 g/mol)	50 mg	0.0003 M
L-Aspartic acid (mw: 133.1 g/mol)	80 mg	0.0006 M
L-Histidine (mw: 155.15 g/mol)	20 mg	0.0001 M
L-Isoleucine (mw: 131.17 g/mol)	50 mg	0.0004 M
L-Leucine (mw: 131.17 g/mol)	100 mg	0.0008 M
L-Lysine (mw: 146.19 g/mol)	50 mg	0.0003 M
L-Methionine (mw: 149.21 g/mol)	20 mg	0.0001 M
L-Phenylalanine (mw: 165.19 g/mol)	50 mg	0.0003 M
L-Threonine (mw: 119.12 g/mol)	100 mg	0.0008 M
L-Tryptophan (mw: 204.23 g/mol)	50 mg	0.0002 M
L-Tyrosine (mw: 181.19 g/mol)	50 mg	0.0003 M
L-Valine (mw: 117.15 g/mol)	140 mg	0.0012 M
Uracil (mw: 112.09 g/mol)	20 mg	0.0002 M

Yeast Two Hybrid (Y2H) Media, Amino Acid Dropout Mixes Preparation Steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 10 mg of Adenine to the solution.
3. Add 50 mg of L-Arginine to the solution.
4. Add 80 mg of L-Aspartic acid to the solution.
5. Add 20 mg of L-Histidine to the solution.
6. Add 50 mg of L-Isoleucine to the solution.
7. Add 100 mg of L-Leucine to the solution.
8. Add 50 mg of L-Lysine to the solution.
9. Add 20 mg of L-Methionine to the solution.
10. Add 50 mg of L-Phenylalanine to the solution.
11. Add 100 mg of L-Threonine to the solution.
12. Add 50 mg of L-Tryptophan to the solution.
13. Add 50 mg of L-Tyrosine to the solution.
14. Add 140 mg of L-Valine to the solution.
15. Add 20 mg of Uracil to the solution.
16. Add distilled water until volume is 1 L.
17. Combine the appropriate ingredients, and mix in a sealed container. Omit the desired dropout component. Haploid yeast requires a single dropout (i.e., -Trp=-T or -Leu=-L); diploid yeast requires double dropouts (i.e., -Leu -Trp=-LT); and selecting for interactions requires triple dropouts (i.e., -Leu-Trp -His=-LTH, plus 3-AT).

Penicillin/Streptomycin/Chloramphenicol Antibiotic Mix Preparation and Recipe

Required components

Component	Amount	Concentration
Penicillin G (mw: 356.37 g/mol)	10 g	0.02806 M
Streptomycin (mw: 728.69 g/mol)	5 g	0.006861 M
Chloramphenicol (mw: 323.13 g/mol)	1 g	0.003094 M

Penicillin/Streptomycin/Chloramphenicol Antibiotic Mix Preparation

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 10 g of Penicillin G to the solution.
3. Add 5 g of Streptomycin to the solution.
4. Add 1 g of Chloramphenicol to the solution.
5. Add distilled water until the volume is 1 L.
6. Filter through a 0.22-µm membrane and store in 5-mL aliquots at ~20°C. For use, add 5 mL of the antibiotic mix to 1 L of Provasoli-enriched seawater (PES).