Team:Osaka/Meeting

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(2009/08/02)
(Lab protocol)
 
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From [[Team:Osaka/Notebook|Notebook]]
From [[Team:Osaka/Notebook|Notebook]]
-
==2009/08/02==
+
=Material and Methods=
-
====Competent cellの作り方====
+
==Preparing CaCl<sub>2</sub>competent 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 OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)
 +
;#When the culture reaches an OD<sub>600</sub> 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 MgCl<sub>2</sub>''' 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 CaCl<sub>2</sub>''' 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 CaCl<sub>2</sub> 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℃
-
;;;;;;# あらかじめ菌を培養しておく。
+
==Transformation of chemical competent cell==
-
;;;;;;# Single colonyを試験管にとり、5mlのLBを加える。
+
;#Thaw competent cells on ice
-
;;;;;;# 37度で一晩おく。
+
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube
-
;;;;;;# 大量に菌が得られたら、40mlのLBを入れた三角フラスコ2つに菌をいれて37度で約2時間Incubate。
+
;#On ice for 30min
-
;;;;;;#分光光度計でOD60=0.3になるまでIncubate。
+
;#Heat shock 42℃ for 2~1min
-
;;;;;;#OD60=0.3になったら、三角フラスコをOn ice 10min (常に道具は冷やした状態をこころがける)
+
;#On ice for 5min
-
;;;;;;#遠心分離管(J.20、サイズに注意、冷やしておく)にピペッターで20mlずつ4本分注。はかりで測って重さを均一にする。
+
;#Add 900 ul LB
-
;;;;;;#8krpm,4℃、roter ID 20、5min で遠心分離。(ローターのサイズにも注意,20 を使う)
+
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)
-
;;;;;;#上澄みをデカンテーション。
+
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)
-
;;;;;;#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。
+
;#Discard 900~800 ul of supernatant
-
;;;;;;#On ice 10min冷蔵室に放置
+
;#Resuspend pellet by pipetting
-
;;;;;;#8krpm,4℃,roterID 20, 5minで遠心分離。
+
;#Plating all cells on the plate with appropriate antibiotics by glass beads
-
;;;;;;#上澄みをデカンテーション。
+
;#Incubate overnight at 37°C
-
;;;;;;#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。
+
;#Watch the plate and find colony
-
;;;;;;#on ice 30min冷蔵室に放置。
+
-
;;;;;;#8krpm,4℃,roterID 20, 5minで遠心分離。
+
-
;;;;;;#上澄みをデカンテーション。(注意深く)
+
-
;;;;;;#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。
+
-
;;;;;;#-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。
+
-
;;;;;;以上です。
+
-
====Transformationの方法====
+
==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)
-
;;;;;#ウェルに15μl milliQ水をいれてピペッティング
+
;#Incubate overnight at 37°C
-
;;;;;#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。)
+
;#Harvest the cells by centrifugation 13krpm, 1min, r.t.
-
;;;;;#コンピ100μlにつき、DNAを2μl加える。
+
;#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
-
;;;;;#30min On ice
+
;#Add '''250 ul Buffer P2''' and inventing the tubes 4-6times
-
;;;;;#2min,42℃でHeat shock
+
;#Add '''350 ul Buffer N3''' and inventing the tubes 4-6times soon after step 5
-
;;;;;#5min on ice
+
;#Centrifuge:15krpm, 10min, 4°C
-
;;;;;#LBを900μl入れる。
+
;#Transfer the supernatants to the Ecospin column (economical..) or QlAprep spin column by pipetting
-
;;;;;#37℃で22min Incubate
+
;#Centrifuge: 13krpm, 1min, r.t.
-
;;;;;#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。
+
;#Reapply the flow-through to column for improving yield
-
;;;;;#プレートに日付、名前をかいて、37℃でIncubate。
+
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through
-
;;;;;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
+
;#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
 +
 
 +
==Restriction Digestion==
 +
For cloning experiments, the final volume should be 20 or 50
</div>
</div>

Latest revision as of 08:02, 11 August 2009

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From Notebook

Contents

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