Team:Macquarie Australia/WetLab/Protocols/Media

From 2014.igem.org

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<li><a href="https://2014.igem.org/Team:Macquarie_Australia/WetLab/Protocols/SDSPAGE">SDS-PAGE</a></li>
<li><a href="https://2014.igem.org/Team:Macquarie_Australia/WetLab/Protocols/SDSPAGE">SDS-PAGE</a></li>
<li><a href="https://2014.igem.org/Team:Macquarie_Australia/WetLab/Protocols/Heat">Heat Shock</a></li>
<li><a href="https://2014.igem.org/Team:Macquarie_Australia/WetLab/Protocols/Heat">Heat Shock</a></li>
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<li><a href="https://2014.igem.org/Team:Macquarie_Australia/WetLab/Protocols/Operons">Induction of Operons</a></li>
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<div class="cont-out">
<div class="cont-out">
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<h3>Media & Plates</h3>
<h3>Media & Plates</h3>
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<p>
 
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Well prepared media are crucial to successful selection and growth of organisms.
 
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<h4>LB AGAR PLATE PREPARATION</h4>
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<p><i>Well prepared media are crucial to successful selection and growth of organisms.</i></p>
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<p>
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<ul style="list-style-type: decimal;">
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<li>Add 15g Bacto agar to 1000mL of LB media and autoclave (121oC for 15mins)</li>
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<li>Add 1000uL of Chloroamphenicol (25mg/mL), Ampicillin (50mg/mL) or Kanamycin (30mg/mL) and mix well before plating out and setting the agar. </li>
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<h4>LB MEDIA</h4>
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<h4>LB AGAR PLATE PREPARATION</h4>
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<b>Ingredients:</b> Tryptone 10g, Yeast extract 5g, NaCl 10g, Milli-Q water to 1000mL
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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).
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<h4>SOC MEDIA (FOR COMPETENT CELLS)</h4>
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<ul style="list-style-type: decimal;">
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<p>
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<li>Add 15g Bacto agar to 1000mL of LB media and autoclave (121<sup>o</sup>C for 15mins).</li>
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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.
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<li>Add 1000uL of Chloroamphenicol (25mg/mL), Ampicillin (50mg/mL) or Kanamycin (30mg/mL) and mix well before plating out and setting the agar. </li>
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</p>
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</ul>
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<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).
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</p>
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<h4>TB BUFFER</h4>
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<img src="https://static.igem.org/mediawiki/2014/3/35/Media.png" height=300 /><img src="https://static.igem.org/mediawiki/2014/d/d7/Media2.png" height=300 />
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<p>
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<p><b>Figure 1.</b> Preparation of LB agar plates.</p>
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<b>Ingredients:</b> 3g PIPES, 10.9g MnCl2-4H2O, 2.0 g CaCl2-2H2O, 18.6 g KCl
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<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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</p>
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<h4>LB MEDIA</h4>
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<ul>
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<li><b>Ingredients:</b> Tryptone 10g, Yeast extract 5g, NaCl 10g, Milli-Q water to 1000mL.</li>
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<li><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<sup>o</sup>C, 15 min, standard liquid cycle). </li>
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</ul>
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<h4>EDTA BUFFER</h4>
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<h4>SOC MEDIA (FOR COMPETENT CELLS)</h4>
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<p>
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<ul>
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<b>Ingredients:</b> 37.22g EDTA solid, 180 mL of water and pH adjusted to 8.0 using 10M NaOH.
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<li><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. </li>
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</p>
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<li><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<sup>o</sup>C, 15 min, standard liquid cycle).</li>
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<p>
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</ul>
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<b>Methods:</b> Components were combined then pH adjusted.
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</p>
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<h4>TAE BUFFER</h4>
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<p>
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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)
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</p>
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<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.
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Latest revision as of 13:09, 17 October 2014

Protocols


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).
  • Add 1000uL of Chloroamphenicol (25mg/mL), Ampicillin (50mg/mL) or Kanamycin (30mg/mL) and mix well before plating out and setting the agar.

Figure 1. Preparation of LB agar plates.

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 (121oC, 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 (121oC, 15 min, standard liquid cycle).

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