Difference between revisions of "Team:Toulouse/Experiments"

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<li> 0.42g MOPS </li>
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<li>1. Streak cells froms frozen stock onto YETM plate. Incubate overnight at 37°C </li>
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<li> 2.21g CaCl2.2H20 </li>
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<li>2. Pick a single fresh colony to inoculate 5mL of YETM medium. Grow over night at 37°C.</li>
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<li value=""> 0.24g RbCl </li>
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<li><b>Do not vortex cells at any time after this point in the procedure</b></li>
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<li> 30g Glycerol </li>
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<li>3. Dilute 1mL of culture into 50mL YETM medium prewarmed to 37°C</li>
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<li> Grow at 37°C for 2hr with agitation </li>
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<li> Volumes can be scaled up 5X and all of the 5mL overnight culture can be used </li>
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</ul>
 
<li> Adjust to pH 6.5 with KOH </li>
 
<li> Adjust to pH 6.5 with KOH </li>
 
<li> Add dH2O to 200mL </li>
 
<li> Add dH2O to 200mL </li>

Revision as of 10:25, 1 September 2015

iGEM Toulouse 2015

Experiments & Protocols


Transformation Protocol: RbCl method

Cloning

Media and solution

YETM 500mL TFB1 200mL TFB2 200mL
  • 2.5g Yeast Extract
  • 10g Tryptone
  • 5g MgSO4.7H2O
  • Adjust pH to 7.5 with KOH
  • For Plates: add 7.5g of Agar
  • 0.59g KOAc
  • 2.42g RbCl
  • 0.29g CaCl2.2H2O
  • 1.98g MnCl2.4H2O
  • Adjust to pH 5.8 with 0.2 M acetic acid
  • Add dH2O to 200mL
  • Filter sterilize
  • Store refrigerated at 4°C
  • 0.42g MOPS
  • 2.21g CaCl2.2H20
  • 0.24g RbCl
  • 30g Glycerol
  • Adjust to pH 6.5 with KOH
  • Add dH2O to 200mL
  • Filter sterilize
  • Store refrigerated at 4°C

Preparation of Competent Cells

  • 1. Streak cells froms frozen stock onto YETM plate. Incubate overnight at 37°C
  • 2. Pick a single fresh colony to inoculate 5mL of YETM medium. Grow over night at 37°C.
  • Do not vortex cells at any time after this point in the procedure
  • 3. Dilute 1mL of culture into 50mL YETM medium prewarmed to 37°C
    • Grow at 37°C for 2hr with agitation
    • Volumes can be scaled up 5X and all of the 5mL overnight culture can be used
  • Adjust to pH 6.5 with KOH
  • Add dH2O to 200mL
  • Filter sterilize
  • Store refrigerated at 4°C

References