Team:HokkaidoU Japan/Notebook/August16

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(→制限酵素の活性チェック)
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=制限酵素の活性チェック=
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=Restriction Enzyme Activity Check=
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==制限酵素処理==
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==Restriction Enzyme Digestion==
<div style="float:left;">
<div style="float:left;">
'''EcoR I, Xba I, Pst I'''
'''EcoR I, Xba I, Pst I'''

Revision as of 08:14, 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
  • EcoR I, Xba I, Pst Iそれぞれ作る

→37℃，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

→37℃，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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