From Notebook

Material and Methods

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
13. Watch the plate and find colony

Plasmid mini prep (QIAprep Spin Miniprep kit)

1. Pick a single colony from transformed cells and inoculate 5 ml LB with the appropriate selective antibiotics (Amp->5 ul, Kan->25 ul)
2. Incubate overnight at 37°C
3. Harvest the cells by centrifugation 13krpm, 1min, r.t.
4. Resuspend pelleted cells in 250 ul Buffer P1. No cell clumps should be visible after resuspension of the pellet. So, pipetting an vortex well!.P1 buffer must be stocked at 4°C
5. Add 250 ul Buffer P2 and inventing the tubes 4-6times
6. Add 350 ul Buffer N3 and inventing the tubes 4-6times soon after step 5
7. Centrifuge:15krpm, 10min, 4°C
8. Transfer the supernatants to the Ecospin column (economical..) or QlAprep spin column by pipetting
9. Centrifuge: 13krpm, 1min, r.t.
10. Reapply the flow-through to column for improving yield
11. Centrifuge: 13krpm, 1min, r.t. and discard the flow-through
12. Add 500 ul Buffer PB in column
13. Centrifuge: 13krpm, 1min, r.t. and discard the flow-through
14. Add 750 ul Buffer PE in column
15. Centrifuge: 13krpm, 1min, r.t. and discard the flow-through
16. Centrifuge: 14krpm, 3min, r.t. to remove residual wash buffer. And then, discard the flow-through
17. Place the column in a clean 1.5 ml tube. Add 100~50 ul Buffer EB or sterile water to the center of each column
18. Keep at r.t. for 3~5min
19. Centrifuge:15krpm, 1min
20. stock -30°C

Restriction Digestion

For cloning experiments, the final volume should be 20 or 50