Difference between revisions of "Team:Toulouse/Experiments"

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<li> 6. Resuspend cells in 10mL of ice-cold TFB1 with gentle re-pipetting. Use chilled glass or plastic pipette </li>
 
<li> 6. Resuspend cells in 10mL of ice-cold TFB1 with gentle re-pipetting. Use chilled glass or plastic pipette </li>
 
<li> 7. Incubate cells on ice for 5min </li>
 
<li> 7. Incubate cells on ice for 5min </li>
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<li> 8. Repeat step 5</li>
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<li> 9. Resuspend cells in 2mL of ice-cold TFB2 with <b>gentle</b> re-pipetting. Use micropipet tip (plastic)</li>
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<li> 10. Incubate cells on ice for 15 minutes </li>
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<li> Cells may be used for transformation or frozen </li>
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<li> To freeze: aliquot cell in 200μL volumes into prechilled 0.5mL microfuge tube (on ice) </li>
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<li> Freeze immediately in liquid nitrogen </li>
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<li> Store cells frozen at -80°C </li>
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</ul>
 
</ul>
 
</ul>
 
</div>
 
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   </main>
 
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Revision as of 10:45, 1 September 2015

iGEM Toulouse 2015

Experiments & Protocols


Transformation Protocol: RbCl method

Cloning

Transformation Protocol: RbCl method

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
  • 4. Transfer culture to sterile 50 mL tube. Chill on ice/water 10-15 minutes
  • 5. Centrifuge for 10 minutes at 2000rpm at 4°C. Immediately aspirate off all of the supernatant
  • Do not allow cells to warm above 4°C at any time in this procedure
  • 6. Resuspend cells in 10mL of ice-cold TFB1 with gentle re-pipetting. Use chilled glass or plastic pipette
  • 7. Incubate cells on ice for 5min
  • 8. Repeat step 5
  • 9. Resuspend cells in 2mL of ice-cold TFB2 with gentle re-pipetting. Use micropipet tip (plastic)
  • 10. Incubate cells on ice for 15 minutes
  • Cells may be used for transformation or frozen
    • To freeze: aliquot cell in 200μL volumes into prechilled 0.5mL microfuge tube (on ice)
    • Freeze immediately in liquid nitrogen
    • Store cells frozen at -80°C

References