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Team:Macquarie Australia/WetLab/Protocols/Media
From 2014.igem.org
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<b>Ingredients:</b> Tryptone 10g, Yeast extract 5g, NaCl 10g, Milli-Q water to 1000mL | <b>Ingredients:</b> Tryptone 10g, Yeast extract 5g, NaCl 10g, Milli-Q water to 1000mL | ||
| + | </p> | ||
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<b>Methods:</b> Dissolved 10g tryptone, 5g yeast extract and 10g NaCl in 800mL Milli-Q water, making use of a magnetic stirrer. Once dissolved, bring volume up to 1 L using Milli-Q water. Autoclave 1L of the solution (121°C, 15 min, standard liquid cycle). | <b>Methods:</b> Dissolved 10g tryptone, 5g yeast extract and 10g NaCl in 800mL Milli-Q water, making use of a magnetic stirrer. Once dissolved, bring volume up to 1 L using Milli-Q water. Autoclave 1L of the solution (121°C, 15 min, standard liquid cycle). | ||
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<b>Ingredients:</b> 10g bacto-tryptone, 2.5g bacto-yeast, 1000µl 5M NaCl, 417µL 3M KCl, 1.205g 20mM Mg2SO4, 805g 20mM D-glucose & 500mL Milli-Q water. | <b>Ingredients:</b> 10g bacto-tryptone, 2.5g bacto-yeast, 1000µl 5M NaCl, 417µL 3M KCl, 1.205g 20mM Mg2SO4, 805g 20mM D-glucose & 500mL Milli-Q water. | ||
| + | </p> | ||
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<b>Methods:</b> The adjusted quantities were combined in a 1 L measuring column with constant stirring and then placed in the autoclave for sterilisation (121°C, 15 min, standard liquid cycle). | <b>Methods:</b> The adjusted quantities were combined in a 1 L measuring column with constant stirring and then placed in the autoclave for sterilisation (121°C, 15 min, standard liquid cycle). | ||
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<b>Ingredients:</b> 3g PIPES, 10.9g MnCl2-4H2O, 2.0 g CaCl2-2H2O, 18.6 g KCl | <b>Ingredients:</b> 3g PIPES, 10.9g MnCl2-4H2O, 2.0 g CaCl2-2H2O, 18.6 g KCl | ||
| + | </p> | ||
| + | <p> | ||
<b>Methods:</b> All components (except for MnCl2-4H2O) were mixed and dissolved in 500 mL of water and pH adjusted to 6.7 with KOH. Then, MnCl2-4H2O, was dissolved in 300 mL of water, mixed and solution adjusted to 1 L. Sterilisation via filtration followed through a pre-rinsed 0.45 µm filter unit and stored at 4°C. | <b>Methods:</b> All components (except for MnCl2-4H2O) were mixed and dissolved in 500 mL of water and pH adjusted to 6.7 with KOH. Then, MnCl2-4H2O, was dissolved in 300 mL of water, mixed and solution adjusted to 1 L. Sterilisation via filtration followed through a pre-rinsed 0.45 µm filter unit and stored at 4°C. | ||
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<b>Ingredients:</b> 37.22g EDTA solid, 180 mL of water and pH adjusted to 8.0 using 10M NaOH. | <b>Ingredients:</b> 37.22g EDTA solid, 180 mL of water and pH adjusted to 8.0 using 10M NaOH. | ||
| + | </p> | ||
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<b>Methods:</b> Components were combined then pH adjusted. | <b>Methods:</b> Components were combined then pH adjusted. | ||
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<b>Ingredients:</b> 121.2g Tris base (dissolved in water) with 28.55mL of glacial acetic acid & 50mL 0.5M EDTA (pH 8.0) | <b>Ingredients:</b> 121.2g Tris base (dissolved in water) with 28.55mL of glacial acetic acid & 50mL 0.5M EDTA (pH 8.0) | ||
| + | </p> | ||
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<b>Methods:</b> A total volume of 500 mL was made up as a 50x stock solution using all components. | <b>Methods:</b> A total volume of 500 mL was made up as a 50x stock solution using all components. | ||
</p> | </p> | ||
Revision as of 05:11, 16 October 2014
Media & Plates
Well prepared media are crucial to successful selection and growth of organisms.
LB AGAR PLATE PREPARATION
- Add 15g Bacto agar to 1000mL of LB media and autoclave (121oC for 15mins)
- 2. Add 1000uL of Chloroamphenicol (25mg/mL), Ampicillin (50mg/mL) or Kanamycin (30mg/mL) and mix well before plating out and setting the agar.
LB MEDIA
Ingredients: Tryptone 10g, Yeast extract 5g, NaCl 10g, Milli-Q water to 1000mL
Methods: Dissolved 10g tryptone, 5g yeast extract and 10g NaCl in 800mL Milli-Q water, making use of a magnetic stirrer. Once dissolved, bring volume up to 1 L using Milli-Q water. Autoclave 1L of the solution (121°C, 15 min, standard liquid cycle).
SOC MEDIA (FOR COMPETENT CELLS)
Ingredients: 10g bacto-tryptone, 2.5g bacto-yeast, 1000µl 5M NaCl, 417µL 3M KCl, 1.205g 20mM Mg2SO4, 805g 20mM D-glucose & 500mL Milli-Q water.
Methods: The adjusted quantities were combined in a 1 L measuring column with constant stirring and then placed in the autoclave for sterilisation (121°C, 15 min, standard liquid cycle).
TB BUFFER
Ingredients: 3g PIPES, 10.9g MnCl2-4H2O, 2.0 g CaCl2-2H2O, 18.6 g KCl
Methods: All components (except for MnCl2-4H2O) were mixed and dissolved in 500 mL of water and pH adjusted to 6.7 with KOH. Then, MnCl2-4H2O, was dissolved in 300 mL of water, mixed and solution adjusted to 1 L. Sterilisation via filtration followed through a pre-rinsed 0.45 µm filter unit and stored at 4°C.
EDTA BUFFER
Ingredients: 37.22g EDTA solid, 180 mL of water and pH adjusted to 8.0 using 10M NaOH.
Methods: Components were combined then pH adjusted.
TAE BUFFER
Ingredients: 121.2g Tris base (dissolved in water) with 28.55mL of glacial acetic acid & 50mL 0.5M EDTA (pH 8.0)
Methods: A total volume of 500 mL was made up as a 50x stock solution using all components.
