Team:HokkaidoU Japan/Notebook/August16

From 2010.igem.org

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==Restriction Enzyme Digestion==
==Restriction Enzyme Digestion==
<div style="float:left;">
<div style="float:left;">
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'''EcoR I, Xba I, Pst I'''
'''EcoR I, Xba I, Pst I'''
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{|style="text-align: center;" class="protocol"
{|style="text-align: center;" class="protocol"
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|style="border-top:1px solid #996"|'''10 uL'''
|style="border-top:1px solid #996"|'''10 uL'''
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* EcoR I, Xba I, Pst Iそれぞれ作る
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→37℃，60 min
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* Made solution for each EcoR I, Xba I, Pst I
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** incubated at 37C for 60 min
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|style="border-top:1px solid #996"|'''10 uL'''
|style="border-top:1px solid #996"|'''10 uL'''
|}
|}
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* pUC119はSpe Iで切れないので，mini prepで得たプラスミドで確かめる
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* pUC119 doesn't have Spe I restriction sites, so we used mini preped plasmid with biobrick insert
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→37℃，60 min
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** incubated at 37C for 60 min
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<div style="clear:both;"></div>
<div style="clear:both;"></div>
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===コントロール===
===コントロール===
* 1 uLのpUC119に1 uLの6x Sample Bufferを加えた
* 1 uLのpUC119に1 uLの6x Sample Bufferを加えた

Revision as of 08:27, 20 September 2010

Notebook

Restriction Enzyme Activity Check

Restriction Enzyme Digestion

EcoR I, Xba I, Pst I

Reagent Amount
pUC119 1 uL
DW 7 uL
10x M Buffer 1 uL
Restriction Enzyme 1 uL
Total 10 uL
  • Made solution for each EcoR I, Xba I, Pst I
    • incubated at 37C for 60 min

Spe I

Reagent Amount
1-1A 1 uL
DW 7 uL
10x M Buffer 1 uL
Restriction Enzyme 1 uL
Total 10 uL
  • pUC119 doesn't have Spe I restriction sites, so we used mini preped plasmid with biobrick insert
    • incubated at 37C for 60 min

コントロール

  • 1 uLのpUC119に1 uLの6x Sample Bufferを加えた
  • 1 uLの1-1Aに1 uLの6x Sample Bufferを加えた

→37℃，60 min

電気泳動

HokkaidoU Japan 20100816a.jpg

制限酵素処理した溶液に2 uLの6x Sample Bufferを加え，電気泳動した

Lane DNA
１ λ/HindIII
２ pUC119 コントロール
３ 1-1A コントロール
４ pUC119 + EcoR I
５ pUC119 + Xba I
６ 1-1A + Spe I
７ pUC119 + Pst I
８ 空き

ベクターの制限酵素処理 (EcoR I, Pst I)

Reagent Amount
pSB1C3 4 uL
DW 10 uL
10x M Buffer 2 uL
BSA 2 uL
EcoR I 1 uL
Pst I 1 uL
Total 20 uL

パーツの制限酵素処理 (Xba I, Pst I)

RBS

Reagent Amount
1-2M 2.5 uL
DW 11.5 uL
10x M Buffer 2 uL
BSA 2 uL
Xba I 1 uL
Pst I 1 uL
Total 20 uL

Terminator

Reagent Amount
1-23L 1.5 uL
DW 12.5 uL
10x M Buffer 2 uL
BSA 2 uL
Xba I 1 uL
Pst I 1 uL
Total 20 uL

パーツの制限酵素処理 (EcoR I, Spe I)

Heat shock promotor

Reagent Amount
3-1E 5 uL
DW 9.7 uL
10x M Buffer 2 uL
BSA 2 uL
EcoR I 1 uL
Spe I 0.3 uL
Total 20 uL

RFP

Reagent Amount
1-18F 5 uL
DW 9.7 uL
10x M Buffer 2 uL
BSA 2 uL
EcoR I 1 uL
Spe I 0.3 uL
Total 20 uL
  • 制限酵素の活性チェックでSpe Iの活性が低かったので，あとから0.7 uL加えた
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