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.