Team:Groningen/Protocols for Lactococcus

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Preparation of cells

  • 10 ml ON culture (SMGG) in 100 ml SMGG
  • Grow to OD600=0.2-0.7
  • Wash three times with 50 ml icecold wash buffer
  • Resuspend in 1 ml wash buffer

Electroporation

  • 1 μl DNA in 40 μl cell-suspension in ice cold cuvette
  • Electroporate at 2.5 kV, 25 μF, 200 Ohm.
  • Add 4 ml SMG17MC
  • Incubate 2 hours (Ery induction=50 ng/ml)
  • Concentrate cells to 0.5 ml.
  • Plate on GSM17- Agar.