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.
  • 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
  • Mix 50-200 ng plasmid DNA with 40 µL of competent cells (always defrost on ice!). Transfer attempt into precooled electroporation cuvette. Incubate on ice for 10 min.
  • Important: DNA must be salt free! Use either directly in ddH₂O eluted plasmids, or (e.g. directly after ligation) desalt the DNA for 30 min on a Millipore filter (MF Membrane Filters).
  • Electroporation: 2500 V for 2 mm cuvettes (1250 V for 1 mm cuvettes), 25 µF, 200 Ω, discharging time should be 3-5 msec. Contacts of the electroporation cuvette must be dry. Remove all air bubbles before electroporation.
  • Immediately add 960 µL liquid LB (or, if available, SOC medium for a higher transformation efficiency) into the cuvette and transfer the content into a 2 ml Eppendorf cup.
  • Incubate at 37 °C and 150 rpm for 1 h. Fix tube in horizontal position with tape.
  • Plate 100 µL of 10^-3 to 10^-6 dilutions on plates with selection pressure
  • Incubate at 37 °C overnight.
• Heat Shock Transformation of Heat Competent Cells
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