Team:Osaka/Meeting

From 2009.igem.org

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====Preparing CaCl<sub>2</sub>competent cell ====
====Preparing CaCl<sub>2</sub>competent cell ====
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<b>#preparation</b>
+
<b>preparation</b>
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;;;;;;#Pick a single colony (strain:Nova Blue) and inoculate 5 ml LB
+
;#Pick a single colony (strain:Nova Blue) and inoculate 5 ml LB
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;;;;;;#Incubate overnight at 37°C  
+
;#Incubate overnight at 37°C  
-
;;;;;;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer
+
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer
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;;;;;;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)
+
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)
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;;;;;;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)
+
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)
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;;;;;;#Keep cells on ice for 10min
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;#On ice for 10min
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;;;;;;#Centrifuge:8krpm, 5min,4°C
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;#Centrifuge:8krpm,5min,4°C
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;;;;;;#Carefully discard supernatant
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;#Discard supernatant
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;;;;;;#0.1M MgCl2溶液(これも使う直前に冷やす）をそれぞれ最初にピペッターで15ml加えてvortex,その後同じものをさらに10ml加える。
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;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)
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;;;;;;#On ice 10min冷蔵室に放置
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;#On ice for 10min
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;;;;;;#8krpm,4℃,roterID 20, 5minで遠心分離。
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;#Centrifuge:8krpm,5min,4℃
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;;;;;;#上澄みをデカンテーション。
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;#Discard supernatant
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;;;;;;#0.1M CaCl2（冷やしておく）をピペッターで25ml加えてvortex、さらに10ml加える。
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;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)
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;;;;;;#on ice 30min冷蔵室に放置。
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;#On ice 30min
-
;;;;;;#8krpm,4℃,roterID 20, 5minで遠心分離。
+
;#Centrifuge:8krpm,5min,4℃
-
;;;;;;#上澄みをデカンテーション。（注意深く）
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;#Discard supernatant carefuly
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;;;;;;#ピペットマンで750μl 0.1M CaCl2、750μl 50％　グリセロール（すべて冷やされているもの）を加えてvortex。
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;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol
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;;;;;;#-80℃に冷やしたペンTubeに遠心分離管の内容物をピペットマンで50μlずつ分注。再び-80℃で保管。
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;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃
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;;;;;;以上です。
+
;#stock at -80℃
 +
 
 +
 
====Transformationの方法====
====Transformationの方法====

Revision as of 05:52, 11 August 2009

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From Notebook

2009/08/02

Preparing CaCl2competent cell

preparation

Pick a single colony (strain
  1. 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の方法

  1. ウェルに15μl milliQ水をいれてピペッティング
  2. 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。）
  3. コンピ100μlにつき、DNAを2μl加える。
  4. 30min On ice
  5. 2min,42℃でHeat shock
  6. 5min on ice
  7. LBを900μl入れる。
  8. 37℃で22min　Incubate
  9. 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。
  10. プレートに日付、名前をかいて、37℃でIncubate。
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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