• LB Medium
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• M9 Minimal Medium
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• B₁₂ Detection Medium
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Cultivation and Transformation

• Cultivation of Escherichia coli, Shimwellia blattae and Salmonella typhimurium TA 100
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• Cultivation of Raoultella planticola
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• Preparation of Electrocompetent Cells

  Material to prepare

  • 2x 5 mL LB
  • 250 mL LB in 1 L Erlenmeyer flask with chicane
  • 500 mL sterile Millipore-H₂O at 4 °C
  • 50 mL sterile glycerol (10 % (w/v)) at 4 °C
  • 30 sterile labeled Eppendorf cups at – 20°C or -80 °C
  • 1x sterile GS3 jar at 4 °C
  • 1x Aquatron at 30 °C
  • 1x Sorvall RC6 centrifuge at 4 °C
  • 1x Universal 320R centrifuge at 4 °C
  • 1 L liquid nitrogen

   

  Day 1

  • Inoculate 2x 5 mL LB (if required with antibiotics) with your strain of interest, either from cryo culture or from agar plate.
  • Incubate overnight at 37 °C and 150 rpm.

   

  Day 2

  • Inoculate 250 mL LB (without antibiotics) with 2 % (5 mL) preculture.
  • Incubate in the Aquatron at 30 °C and 160 rpm until OD₆₀₀ of 0.5-0.8.
  • Time for E. coli DH5α: approx. 4 h.
  • Check the culture via microscope for contaminations.

  All liquids and containers must be cooled on ice. The major task of cell preparation is the removal of salts. In case of some strains like pLys-strains, the pellets must be resuspended very carefully.

  • Let the cells (OD₆₀₀ of 0.5-0.8) cool down in ice water for 10-20 min. All further steps are performed under cool conditions.
  • Decant the cells in sterile GS3 jar and centrifuge at 4 °C and 5000 rpm (4230 g) for 5-10 min.
  • Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 250 mL sterile 4 °C ddH₂O.
  • Centrifuge the GS3 jar at 4 °C and 5000 rpm (4230 g)for 5-10 min.
  • Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 250 mL sterile 4 °C ddH₂O.
  • Centrifuge the GS3 jar at 4 °C and 5000 rpm (4230 g)for 5-10 min.
  • Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 10 mL sterile 4 °C glycerol (10 % (w/v)). Afterwards, transfer the culture into a 50 mL Falcon tube.
  • Centrifuge the Falcon tube at 4 °C and 6200 rpm (4230 g) for 5-10 min.
  • Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 10 mL sterile 4 °C glycerol (10 % (w/v)).
  • Centrifuge the Falcon tube at 4 °C and 6200 rpm (4230 g) for 5-10 min.
  • Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 500 µL sterile 4 °C glycerol (10 % (w/v)).
  • Aliquot the cells into the Eppendorf cups (40 µL per Eppendorf cup). While aliquoting, filled Eppendorf cups must be frozen directly in liquid nitrogen.
  • Store the filled Eppendorf cups in a cryobox at -80 °C.
• Transformation Electroporation of Electrocompetent Cells
  Curabitur ullamcorper ultricies nisi. Nam eget dui. Etiam rhoncus. Maecenas tempus, tellus eget condimentum rhoncus, sem quam semper libero, sit amet adipiscing sem neque sed ipsum. Nam quam nunc, blandit vel, luctus pulvinar, hendrerit id, lorem. Maecenas nec odio et ante tincidunt tempus. Donec vitae sapien ut libero venenatis faucibus. Nullam quis ante. Etiam sit amet orci eget eros faucibus tincidunt. Duis leo. Sed fringilla mauris sit amet nibh. Donec sodales sagittis magna. Sed consequat, leo eget bibendum sodales, augue velit cursus nunc,
• Heat Shock Transformation of Heat Competent Cells
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