Team:HokkaidoU Japan/Notebook/September27

From 2010.igem.org

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(→ようやくSalmonella TyphimuriumのBACクローンが届いた)
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** Codes Type 3 Secretion Aparatus  
** Codes Type 3 Secretion Aparatus  
* BAC clones of S. Typhimurium, B_STM02P01　'''SGSC4014'''　837453-941863
* BAC clones of S. Typhimurium, B_STM02P01　'''SGSC4014'''　837453-941863
-
** 分泌シグナルをコードしている領域を含む
+
** Codes Secretion Signal
----
----
-
==BAC cloneの培養==
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==Culture of the BAC clone==
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===25 ug/mL Chloramphenicol培地の作製===
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* made 25 ug/mL Chloramphenicol LB Agar plate
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* 25 ug/mL Chloramphenicol LB寒天培地を25枚作製
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* made 25 ug/mL Chloramphenicol LB medium
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* 25 ug/mL Chloramphenicol LB液体培地を2本作製
+
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===前培養===
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===Preculture===
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* 培地を作製している間，送られてきたそれぞれの菌をAgar stabからLB液体培地に移して約２時間前培養した
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* while preparing the plate, cultibated each of the clones in LB medium for 2 hrs
 +
 
 +
===Culture===
 +
* plated the precultured BAC clones into LBC plate respectively
-
===培養===
 
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* 前培養していたLBをそれぞれ２枚のプレートへ展開した
 
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** 片方は菌が薄くなるよう白金耳で展開し，もう片方には約300 uLを展開した
 
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* 前培養していたLB約1.5 mLを25 ug/mL Chloramphenicol LB液体培地へ加えた
 
-
<!--** 今気づいたけど，これだと25 ug/mLが薄まってしまうね-->
 
----
----
==Electroporation==
==Electroporation==
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===精製===
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===Purification===
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木曜日に作った1 ng/uLのBAC solutionの塩濃度を下げるため，Microcon YM-10で精製した
+
In order to decrease salt concentration, purified 1 ng/uL of BAC solution which has been prepared on Thursday using Microcon YM-10
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* DNA solution 250 uLとDW450 uLをMicrocon YM-10のカラムに入れ，14,000rpm @4Cで１時間遠心した
+
 
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* 新しいチューブにカラムを逆さにつけて，14,000rpm @4Cで１０分間遠心した
+
-
* 30 uL回収できたため，220 uLのDWを加え，元の量にした
+
-
===エレポ===
+
===Electroporation===
-
精製したDNA solution 1 uLを用いて，DH5aとMG1655のエレポを行った
+
electroporated DH5a and MG1655 using 1 uL of purified DNA solution
* MG1655: 2.5 kV: 5.4 ms
* MG1655: 2.5 kV: 5.4 ms
* DH5a: 2.49 kV: 5.7 ms
* DH5a: 2.49 kV: 5.7 ms
-
SOB 200 uLを加えてラウンドチューブに移して浸透しながら2時間培養し，Ampicillin培地に展開した
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* added 200 uL of SOB and transfered into round tube
 +
* incubated at 37C for 2 hrs
 +
* plated onto LBA

Revision as of 12:44, 26 October 2010

Notebook

Finally Salmonella Typhimurium BAC library clones have arrived!

  • BAC clones of S. Typhimurium, B_STM07H21 SGSC4024 1464540-1562427
    • Codes Type 3 Secretion Aparatus
  • BAC clones of S. Typhimurium, B_STM02P01　SGSC4014　837453-941863
    • Codes Secretion Signal


Culture of the BAC clone

  • made 25 ug/mL Chloramphenicol LB Agar plate
  • made 25 ug/mL Chloramphenicol LB medium

Preculture

  • while preparing the plate, cultibated each of the clones in LB medium for 2 hrs

Culture

  • plated the precultured BAC clones into LBC plate respectively


Electroporation

Purification

In order to decrease salt concentration, purified 1 ng/uL of BAC solution which has been prepared on Thursday using Microcon YM-10


Electroporation

electroporated DH5a and MG1655 using 1 uL of purified DNA solution

  • MG1655: 2.5 kV: 5.4 ms
  • DH5a: 2.49 kV: 5.7 ms
  • added 200 uL of SOB and transfered into round tube
  • incubated at 37C for 2 hrs
  • plated onto LBA
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