Team:TzuChiU Formosa/Protocol

• Home
• Projects
  • Overview
  • Protocol
  • Result
  • Safety
• Team
• Human Practices
• Notebook
  • Summary
  • Week 1
  • Week 2
  • Week 3
  • Week 4
  • Week 5
  • Week 6
  • Week 7
  • Week 8
  • Week 9
• Partners
• Judging Form
• Parts

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)

Competent CFU test - Transformation-E.coli

Test competent cell CFU the day before

1. Defrost the 200 ul competent cell on ice.
2. Add 10 ng plasmid into the 200 ul competent cell.
3. Place it on ice for 30 minutes
4. Heat shock in water bath at 42℃ for 1 and a half minute then place it on ice for 5 minutes.
5. Add in 800 ul LB and place it in the 37℃ incubator for 1 hour.
6. Test the frequency
7. Incubate at 37℃for 12~16 hours
8. Select colony and check via quick- screening.