From Notebook

Lab protocol

Preparing CaCl₂competent cell

1. Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB
2. Incubate overnight at 37°C
3. Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer
4. Incubate culture at 37°C on a shaker up to OD₆₀₀=0.3~0.5 (Measure OD value first 1hr and each 30min)
5. When the culture reaches an OD₆₀₀ between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)
6. On ice for 10min
7. Centrifuge:8krpm,5min,4°C
8. Discard supernatant
9. Resuspend each pellet in 20 ml chilled 0.1 M MgCl₂ and add further 15 ml 0.1 M MgCl (total 45 ml)
10. On ice for 10min
11. Centrifuge:8krpm,5min,4℃
12. Discard supernatant
13. Resuspend each pellet in 20 ml chilled 0.1 M CaCl₂ and add further 15 ml 0.1 M MgCl (total 45 ml)
14. On ice 30min
15. Centrifuge:8krpm,5min,4℃
16. Discard supernatant carefuly
17. Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl₂ and 750 µl pre-chilled 50%(v/v) Glycerol
18. Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃
19. stock at -80℃

Transformation of chemical competent cell

1. Thaw competent cells on ice
2. Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube
3. On ice for 30min
4. Heat shock 42℃ for 2~1min
5. On ice for 5min
6. Add 900 ul LB
7. Incubate at 37℃ for 1hour (During this, warm plates at 42℃)
8. Harvest cells by centrifuge (14krpm, 1min, r.t.)
9. Discard 900~800 ul of supernatant
10. Resuspend pellet by pipetting
11. Plating all cells on the plate with appropriate antibiotics by glass beads
12. Incubate overnight at 37°C