Team:Osaka/Meeting

From 2009.igem.org

(Difference between revisions)
(Preparing CaCl2competent cell)
(2009/08/02)
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;#stock at -80℃
;#stock at -80℃
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====Transformationの方法====
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====Transformation of chemical competent cell====
   
   
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;;;;;#ウェルに15μl milliQ水をいれてピペッティング
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;#Thaw competent cells on ice
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;;;;;#エッペンTubeに回収、ナンバリングを忘れないように。(DNAプレートに書いてある数とアルファベット、その上にプレートナンバーを書く。)
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;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube
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;;;;;#コンピ100μlにつき、DNAを2μl加える。
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;#On ice for 30min
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;;;;;#30min On ice
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;#Heat shock 42℃ for 2~1min
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;;;;;#2min,42℃でHeat shock
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;#On ice for 5min
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;;;;;#5min on ice
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;#Add 900 ul LB
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;;;;;#LBを900μl入れる。
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;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)
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;;;;;#37℃で22min Incubate
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;#Harvest cells by centrifuge (14krpm, 1min, r.t.)
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;;;;;#platingを行う。プレートにエッペンTubeの内容物をあけ、autoclaveされたビーズをいれてふる。
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;#Discard 900~800 ul of supernatant
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;;;;;#プレートに日付、名前をかいて、37℃でIncubate。
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;#Resuspend pellet by pipetting
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;;;;;以上です。不明な点はのりさん、とりっぴー、ただしまでどうぞ。文責 中村 匡
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;#Plating all cells on the plate with appropriate antibiotics by glass beads
-
 
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;#Incubate overnight at 37°C
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</div>
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Revision as of 06:20, 11 August 2009

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From Notebook

2009/08/02

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