• ELECTROPORATING E. COLI

• 点击：    作者：51protocol收集   来源： 日期：2007-03-25    本站论坛

1. O/N (5mL E. coli in LB -->1mL into 200mL LB/37^oC
   
2. Grow 2-3H to A₆₆₀ ~0.3-0.4
   
3. Chill cells 10'/ice Spin down 10', 5K, 4^oC
   
4. Wash 1X 200mL 10% glycerol (sterile) 40C; Spin down 10', 5K, 4^oC
   
5. Wash 1X 40 mL 10% glycerol --> transfer to S34 tubes & spin down 5', 5K, 4^oC
   **wash means complete resuspention to remove all salt
   
6. Invert tube to drain SN ~5sec (soft plt). with p200, resuspend cells in remaining SN & transfer to eppendorf. Wash SS34 tube with another 100ul 10% glycerol and pool with cells. Vf ~300-500 ul. Use ~ 50 ul cells/electroporation.
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7. Turn on machine (back) -->set 2.5kV, 25 uF. Pulse controller to 200 ohms (wide 0.2cm cuvette)
   
8. 1-2 ul ligation/ 50 ul cells (eppendorf). Chill cuvettes.
   
9. Transfer cells & DNA to cold 0.2cm cuvette
   
10. IMMEDIATELY electroporate @ 2.5kV, 25 uF, 200 ohms (expect time constant 3.5-5msec)
    
11. IMMEDIATELY add 1mL cold SOC. Transfer to eppendorf.
    
12. 37^oC/60'
    
13. Plate 50-200 ul cells (50 ~ 200-300 colonies)
    

Dr. DE Koshland, Carnegie Institution of Washington