Team:Osaka/Meeting
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From Notebook
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Lab protocol
Preparing CaCl2competent cell
- Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB
- Incubate overnight at 37°C
- Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer
- Incubate culture at 37°C on a shaker up to OD600=0.3~0.5 (Measure OD value first 1hr and each 30min)
- When the culture reaches an OD600 between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)
- On ice for 10min
- Centrifuge:8krpm,5min,4°C
- Discard supernatant
- Resuspend each pellet in 20 ml chilled 0.1 M MgCl2 and add further 15 ml 0.1 M MgCl (total 45 ml)
- On ice for 10min
- Centrifuge:8krpm,5min,4℃
- Discard supernatant
- Resuspend each pellet in 20 ml chilled 0.1 M CaCl2 and add further 15 ml 0.1 M MgCl (total 45 ml)
- On ice 30min
- Centrifuge:8krpm,5min,4℃
- Discard supernatant carefuly
- Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl2 and 750 µl pre-chilled 50%(v/v) Glycerol
- Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃
- stock at -80℃
Transformation of chemical competent cell
- Thaw competent cells on ice
- Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube
- On ice for 30min
- Heat shock 42℃ for 2~1min
- On ice for 5min
- Add 900 ul LB
- Incubate at 37℃ for 1hour (During this, warm plates at 42℃)
- Harvest cells by centrifuge (14krpm, 1min, r.t.)
- Discard 900~800 ul of supernatant
- Resuspend pellet by pipetting
- Plating all cells on the plate with appropriate antibiotics by glass beads
- Incubate overnight at 37°C
- Watch the plate and find colony
Plasmid mini prep (QIAprep Spin Miniprep kit)
- Pick a single colony from transformed cells and inoculate 5 ml LB with the appropriate selective antibiotics (Amp->5 ul, Kan->25 ul)
- Incubate overnight at 37°C
- Harvest the cells by centrifugation 13krpm, 1min, r.t.
- 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
- Add 250 ul Buffer P2 and inventing the tubes 4-6times
- Add 350 ul Buffer N3 and inventing the tubes 4-6times soon after step 5
- Centrifuge:15krpm, 10min, 4°C
- Transfer the supernatants to the Ecospin column (economical..) or QlAprep spin column by pipetting
- Centrifuge: 13krpm, 1min, r.t.
- Reapply the flow-through to column for improving yield
- Centrifuge: 13krpm, 1min, r.t. and discard the flow-through
- Add 500 ul Buffer PB in column
- Centrifuge: 13krpm, 1min, r.t. and discard the flow-through
- Add 750 ul Buffer PE in column
- Centrifuge: 13krpm, 1min, r.t. and discard the flow-through
- Centrifuge: 14krpm, 3min, r.t. to remove residual wash buffer. And then, discard the flow-through
- 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
- Keep at r.t. for 3~5min
- Centrifuge:15krpm, 1min
- stock -30°C