Team:Wageningen UR/Protocol/StartupHepB

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Next: [[Team:Wageningen_UR/Protocol/DialysisHepB|HepB dialysis]]

Revision as of 14:21, 24 October 2012


Reagents & Materials

For a 50 mL culture:

reagents:

  • 60 mL of LB
  • kanamycin
  • 0.25 mL of IPTG stock solution 250 mM
  • MQ-water

materials:

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

Procedure

  1. Prepare a Erlenmeyer flask with 50mL 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 (0.25 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 ± 10 mL washing buffer 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: HepB dialysis

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