Quint Lab:preparing electrocompetent A.rhizogenes cells + electroporation

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preparing electrocompetent A. rhizogenis cells

  1. Inoculate 5ml overnight culture (LB medium + strep) with A. rhizogenis. Grow at 28°C (180 rpm) to an OD = 1 (takes ~ 12 hours).
  2. Transfer ON culture into 200 ml preheated LB medium (+ strep). Allow cells to grow at 28°C (180 rpm) to an OD 0,5-0,6 (takes at least 4-5-h).
  3. Put cell culture on ice for 15 min.
  4. Transfer cells culture into 4 prechilled Falcon tubes (50 ml).
  5. centrifugation (10 min, 3000g, 4°C)
  6. Pour off media and resuspend cells with 40 ml cold and sterile water on ice.
  7. centrifugation (10 min, 3000g, 4°C)
  8. Pour off media and resuspend cells with 20 ml cold and sterile water on ice.
  9. centrifugation (10 min, 3000g, 4°C)
  10. Pour off media and resuspend cells with 10 ml cold and sterile 10% glycerol on ice.
  11. Coalesce the 4 fractions.
  12. centrifugation (10 min, 3000g, 4°C)
  13. Pour off media and resuspend cells with 1 ml cold and sterile 10% glycerol on ice.
  14. Devide into 50 µl aliquots and freeze them immediately in liquid nitrogen. Store at -80°C.

elctroporation

  1. Thaw competent cells on ice.
  2. Prechill cuvette (2mm electrode gap).
  3. Pipette 2-5 µl DNA (ca. 150 ng/µl) into the cuvette, add 50 µl competent cells.
  4. electroporation (programm Agr/Ec2).
  5. Add 1ml LB medium and transfer the cells into a 12 ml Falcon tube, fill up to 3 ml with LB medium.
  6. Incubate culture for 3 hours at 28°C (180 rpm).
  7. Directly remove 5 µl and 50 µl from the culture and plate the aliquots on LB-agar plates (+ strep and other appropriate antibiotics).
  8. Incubate plates at 28°C for at least 2 days.