- Vitamin Solution (PES30)
- Vitamin Solution (IMR)
- 1M Tris
- Trace Metal Solution (IMR)
- Trace Metal Solution A5+Co
- TESC
- TE Buffer
- TBE
- TAE
- 10% SDS
- 25% Ringer Solution
- -Pi Stock
- +Pi Stock
- PES Stock Solution
- PCI Solution (25:24:1)
- Micronutrient Solution
- Microelement Stock
- Iron Solution
- Fe-EDTA (A&A)
- 0.5M EDTA
- CI Solution (24:1)
- ATCC Culture Medium 929
- ASNIII
- Allen and Arnon Medium (Plates)
- Allen and Arnon Medium (Liquid)
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
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 4oC
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 4oC
| 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 4oC
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
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
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 4oC
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 4o
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 4oC
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
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
-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 4oC, shake well before use, do not autoclave
+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 4oC
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 4oC in 10 mL aliquots
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 4oC
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
Microelement Stock
| Fe-EDTA solution | 80 mL |
| MnCl2•4H2O | 180 mg |
| MoO3 (85% purity) | 18 mg |
| or Na2Mo4•2H2O (99% purity) |
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)
Iron Solution
| FeNH4(SO4)2•10H2O | 351 mg |
| 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
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)
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 4oC
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 4oC
ATCC Culture Medium 929
| Ammonium Sulfate | 1.32 mg |
| MgSO4•7H2O | 380 mg |
| CaCl2•2H2O | 20 mg |
| MnCl2•4H2O, 50.5 mM solution | 20 µL |
| Na2MoO4•2H2O, 41.3 mM solution | 10 µL |
| CoCl2•6H2O, 1.7 mM solution | 5 µL |
| ZnSO4•7H2O, 34.8 mM solution | 10 µL |
| K2HPO4 | 87 mg |
| Phenol red 0.5% | 250 µL |
| Iron Solution | 250 µL |
| 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 7.5 with 0.5M K2CO3
- Autoclave for 20 minutes
pH: 7.5
ASNIII
| NaCl | 25 g |
| MgCl2•6H2O | 2 g |
| KCl | 500 mg |
| NaNO3 | 750 mg |
| K2HPO4•3H2O | 20 mg |
| MgSO4•7H2O | 3.5 g |
| CaCl2•2H2O | 500 mg |
| Citric Acid | 3 mg |
| Ferric Ammonium Citrate | 3 mg |
| EDTA (disodium magnesium) | 0.5 mg |
| Na2CO3 | 20 mg |
| Trace Metal Solution | 1 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
- Autoclave for 20 minutes
pH: 7.5
Allen and Arnon Medium (Plates)
| -Pi stock | 25 mL |
| +Pi stock | 6.25 mL |
| Agar | 10 g |
| Streptomycin Stock (1 mg/mL) | 1 mL |
| Neomycin Stock (25 mg/mL) | 1 mL |
| Milli-Q Water | ~1 L |
- Measure out approximately 300 mL Milli-Q
- Shake -Pi stock
- Add both stocks to water
- Bring volume up to 1 L with Milli-Q
- Check pH. If less than 7.8, make fresh +Pi
- Add agar
- Autoclave for 20 minutes
- Add antibiotics
Allen and Arnon Medium (Liquid)
| -Pi stock | 6.30 mL |
| +Pi stock | 3.10 mL |
| Streptomycin Stock (1 mg/mL) | 1 mL |
| Neomycin Stock (25 mg/mL) | 1 mL |
| Milli-Q Water | ~1 L |
- Measure out approximately 300 mL Milli-Q
- Shake -Pi stock
- Add both stocks to water
- Bring volume up to 1 L with Milli-Q
- Check pH. If less than 7.8, make fresh +Pi
- Autoclave for 20 minutes
- Add antibiotics