preparing electrocompetent A. rhizogenis cells
- Inoculate 5ml overnight culture (LB medium + strep) with A. rhizogenis. Grow at 28°C (180 rpm) to an OD = 1 (takes ~ 12 hours).
- 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).
- Put cell culture on ice for 15 min.
- Transfer cells culture into 4 prechilled Falcon tubes (50 ml).
- centrifugation (10 min, 3000g, 4°C)
- Pour off media and resuspend cells with 40 ml cold and sterile water on ice.
- centrifugation (10 min, 3000g, 4°C)
- Pour off media and resuspend cells with 20 ml cold and sterile water on ice.
- centrifugation (10 min, 3000g, 4°C)
- Pour off media and resuspend cells with 10 ml cold and sterile 10% glycerol on ice.
- Coalesce the 4 fractions.
- centrifugation (10 min, 3000g, 4°C)
- Pour off media and resuspend cells with 1 ml cold and sterile 10% glycerol on ice.
- Devide into 50 µl aliquots and freeze them immediately in liquid nitrogen. Store at -80°C.
elctroporation
- Thaw competent cells on ice.
- Prechill cuvette (2mm electrode gap).
- Pipette 2-5 µl DNA (ca. 150 ng/µl) into the cuvette, add 50 µl competent cells.
- electroporation (programm Agr/Ec2).
- Add 1ml LB medium and transfer the cells into a 12 ml Falcon tube, fill up to 3 ml with LB medium.
- Incubate culture for 3 hours at 28°C (180 rpm).
- Directly remove 5 µl and 50 µl from the culture and plate the aliquots on LB-agar plates (+ strep and other appropriate antibiotics).
- Incubate plates at 28°C for at least 2 days.
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