Team:Wageningen UR/Protocol/StartupCCMV

From 2012.igem.org

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== Reagents & Materials ==
== Reagents & Materials ==
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 +
For a 100 mL batch:
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 +
Reagents:
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<ul>
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<li>110 mL of LB</li>
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<li>kanamycin</li>
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<li>0.5 mL of IPTG stock solution 250 mM</li>
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<li>MQ-water</li>
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</ul>
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Materials:
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<ul>
 +
<li>Pipettes and pipettes points</li>
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<li>500 ml Erlenmeyer</li>
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<li>Greiner tubes</li>
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<li>Freezer</li>
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<li>Centrifuge for Greiner tubes</li>
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</ul>
== Procedure ==
== Procedure ==
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<li>Induce with '''1.25 mM IPTG''' (0.5 mL of a 250 mM stock)</li>
<li>Induce with '''1.25 mM IPTG''' (0.5 mL of a 250 mM stock)</li>
<li>Incubate at 37°C for 4 h</li>
<li>Incubate at 37°C for 4 h</li>
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<li>Centrifuge the cells in two 500 mL flask in the Sorvall centrifuge at 5000 rpm for 15 minutes</li>
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<li>Centrifuge the cells in 50ml greiner tubes  at 4700 rpm for 18 minutes</li>
<li>Decant the supernatant and resuspend the pellets in ± 20 mL mQ water each</li>
<li>Decant the supernatant and resuspend the pellets in ± 20 mL mQ water each</li>
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<li>Combine the resuspended cells in a 50 mL Greiner  tube and centrifuge at 5000 rpm for 15 minutes</li>
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<li>Centrifuge again at 4700 rpm for 18 minutes</li>
<li>Decant the supernatant and centrifuge for another minute</li>
<li>Decant the supernatant and centrifuge for another minute</li>
<li>Pipette any liquid to clear the tube of supernatant</li>
<li>Pipette any liquid to clear the tube of supernatant</li>
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[[Team:Wageningen_UR/Protocol/DialysisCCMV|Continue further for CCMV formation]]
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Next: [[Team:Wageningen_UR/Protocol/DialysisCCMV|CCMV dialysis]]

Latest revision as of 14:23, 24 October 2012


Growing Culture

Reagents & Materials

For a 100 mL batch:

Reagents:

  • 110 mL of LB
  • kanamycin
  • 0.5 mL of IPTG stock solution 250 mM
  • MQ-water

Materials:

  • Pipettes and pipettes points
  • 500 ml Erlenmeyer
  • Greiner tubes
  • Freezer
  • Centrifuge for Greiner tubes

Procedure

  1. Prepare a Erlenmeyer flask with 0.1 L of LB-medium and keep it at 37°C
  2. Pick E.coli BL21 from a plate and grow them over night in a 10 mL LB culture containing 50mg/L Kanamycin at 37°C
  3. Inoculate the large flask with 1 mL of the overnight culture and grow at 37°C for 2.5h until the OD600 reaches approx. 0.6
  4. Induce with 1.25 mM IPTG (0.5 mL of a 250 mM stock)
  5. Incubate at 37°C for 4 h
  6. Centrifuge the cells in 50ml greiner tubes at 4700 rpm for 18 minutes
  7. Decant the supernatant and resuspend the pellets in ± 20 mL mQ water each
  8. Centrifuge again at 4700 rpm for 18 minutes
  9. Decant the supernatant and centrifuge for another minute
  10. Pipette any liquid to clear the tube of supernatant
  11. (possible to freeze the cells to -20°C at this point)


Next: CCMV dialysis

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