Team:TzuChiU Formosa/Protocol

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Competent cell

1. Culture DH5-alpha in a glass test tube with 3CC LB at 37°C for 12 hours.
2. Add 200 ul bacterium into a 500cc conical flask with 50 ml LB inside. Culture at 37°C until OD600 reaches 0.2
3. 3. Distribute the bacterium from the conical flask into a 50cc Centrifuge and centrifuge at 4000rpm, 15 minutes at 4 °C
4. Remove supernatant and add in 16 ml CaCl2 (100mM)
5. Centrifuge at 4000rpm, 15 minutes at 4 °C
6. Remove supernatant and add 3 ml CaCl2 (100mM)
7. Centrifuge at 4000rpm, 15 minutes at 4 °C
8. Remove supernatant and add 3 ml CaCl2 (100mM)
9. Place on ice and put in 4 °C cold room for an hour.
10. Cut tip and add 1 ml Glycerol (Takes up 25% of the total volume)
11. Transfer 200 ul into each eppendorf ( This step should be done quickly and taken to -80°C ASAP)
    * Can use dry ice or ice with alcohol.
12. Plating the next day to test CFU (colony frequency unit)