Preparing electrocompetent A.

rhizogenis cells
1. Inoculate 5ml overnight culture (LB medium + strep) with A. rhizogenis. Grow at 28C (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 28C (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, 4C). 6. Pour off media and resuspend cells with 40 ml cold and sterile water on ice. 7. Centrifugation (10 min, 3000g, 4C). 8. Pour off media and resuspend cells with 20 ml cold and sterile water on ice. 9. Centrifugation (10 min, 3000g, 4C). 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, 4C). 13. Pour off media and resuspend cells with 1 ml cold and sterile 10% glycerol on ice. 14. Devide into 50 l aliquots and freeze them immediately in liquid nitrogen. Store at -80C.

Electroporation
1. Thaw competent cells on ice. 2. Prechill cuvette (2mm electrode gap). 3. Pipette 2-5 l DNA (ca. 150 ng/l) into the cuvette, add 50 l 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 28C (180 rpm). 7. Directly remove 5 l and 50 l from the culture and plate the aliquots on LB-agar plates (+ strep and other appropriate antibiotics). 8. Incubate plates at 28C for at least 2 days.

Tham kho:

http://openwetware.org/wiki/Quint_Lab:preparing_electrocompetent_A.rhizogenes_cells_%2B_e lectroporation