Team:HokkaidoU Japan/Notebook/September6

From 2010.igem.org

(Difference between revisions)
(→1-3A由来のpSB1C3と1-5A（RFP reporter）)
(→1-3A由来のpSB1C3と1-5A（RFP reporter）)
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=1-3A由来のpSB1C3と1-5A（RFP reporter）=
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=Ligation of pSB1C3 PCRed from 1-3 and 1-5A（RFP reporter）=
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* 制限酵素処理をする前にプライマーなどを確実に除くため，PCR productsの[[ゲル抽出]]を行った
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* Purified through gel extraction to remove primers
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* 次の組成で制限酵素処理を行った
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* Digested as listed in the table below
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* これらをゲル抽し，25 uLのDNase free water
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* Extracted from filter with  25 uL, 50 uL DNase free water
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* この時点でそれぞれ22.56 ng/uL，45.12 ng/uLとなっていると見積もられる
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* At this point solution volumes were 22.56 ng/uL，45.12 ng/uL acordingly
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* pSB1C3 2 uL, RFP 3 uLにMighty Mix 5 uLとT4 ligase 0.5 uLを加え，Ligationした
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* Added 5 uL Mighty Mix and 0.5 uL T4 ligase to pSB1C3 2 uL, and RFP 3 uL
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* 50 uLのコンピテントセルをつかいTransformationした
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* Ligated
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* Transformed to 50 uL of competent cells

Revision as of 17:42, 27 September 2010

Notebook

Ligation of pSB1C3 PCRed from 1-3 and 1-5A（RFP reporter）

  • Purified through gel extraction to remove primers
  • Digested as listed in the table below
Reagent Amount
RFP (1-5A) 0.75 uL
DW 34.3 uL
0.1% BSA 5 uL
10x M Buffer 5 uL
EcoR I 2.5 uL
Pst I 2.5 uL
Total 50 uL
Reagent Amount
pSB1C3 0.75 uL
DW 34.3 uL
0.1% BSA 5 uL
10x M Buffer 5 uL
EcoR I 2.5 uL
Pst I 2.5 uL
Total 50 uL
Reagent Amount
RFP (1-5A) 0.75 uL
DW 34.3 uL
0.1% BSA 5 uL
10x H Buffer 5 uL
EcoR I 2.5 uL
Pst I 2.5 uL
Total 50 uL
Reagent Amount
pSB1C3 0.75 uL
DW 34.3 uL
0.1% BSA 5 uL
10x H Buffer 5 uL
EcoR I 2.5 uL
Pst I 2.5 uL
Total 50 uL
  • Extracted from filter with 25 uL, 50 uL DNase free water
  • At this point solution volumes were 22.56 ng/uL，45.12 ng/uL acordingly
  • Added 5 uL Mighty Mix and 0.5 uL T4 ligase to pSB1C3 2 uL, and RFP 3 uL
  • Ligated
  • Transformed to 50 uL of competent cells
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