Team:Osaka/Meeting
From 2009.igem.org
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(→Preparing CaCl2competent cell) |
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;#stock 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 | |
- | + |
Revision as of 06:20, 11 August 2009
From Notebook
2009/08/02
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