Team:HokkaidoU Japan/Notebook/August30

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(Difference between revisions)
(→pUC119のEcoR I, Pst I digestion)
(→pUC119のEcoR I, Pst I digestion)
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=pUC119のEcoR I, Pst I digestion=
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=Digestion of pUC119 by EcoR I, Pst I=
{|border="1" style="text-align:center;" class="protocol"
{|border="1" style="text-align:center;" class="protocol"
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→Electrophoresed 2 uL for confirmation
→Electrophoresed 2 uL for confirmation
* There were no bands, forgot to add DNA :(
* There were no bands, forgot to add DNA :(
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* 残った18 uLにDW 1 uLとDNAを加えて
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* Reused the remaining 18 uL of digestion solution by adding 1 uL of ADW and 1uL of DNA
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→37℃, 60 min<br>
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→Incubated at 37C for 60 min<br>
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→2 uLを電気泳動で確認（+ 0.4 uL 6x SB）
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→Electrophoresed 2 uL of each solution（+ 0.4 uL 6x SB）
===Electrophoresis===
===Electrophoresis===
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[[Image:HokkaidoU Japan 20100830b.jpg‎|200px|right|thumb|]]
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[[Image:HokkaidoU Japan 20100830b.jpg‎|200px|right|thumb|Electrophoresis of digestion products]]
 +
 
{| class="protocol"
{| class="protocol"
|'''Lane'''
|'''Lane'''
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|-
|-
|2
|2
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|λ/''Hin''d III, EcoR I（4 uL使用）
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|[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png Lambda/''Hin''dIII,　EcoR I]（4 uL used）
|-
|-
|3
|3
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|切ってない
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|Undigested
|-
|-
|4
|4
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|-
|-
|7
|7
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|古いEcoR I
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|EcoR I (used old enzyme to check it's activity)
|}
|}
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* レーン３ではモノマー，ダイマー，トライマーなどのプラスミドが見られる．
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* In lane 3 monomers, dimers and trimers of plasmid were visible ．
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* レーン４～７ではこれらが切れているのが分かる
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* From lanes 4 through 7 it's visible that DNA digestion wasn't satisfactory
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** リニアになったので，モノマーの環状DNA（超コイル）より流れにくい
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** Because plasmid became linear it's was slower than super-coiled one's
==ラオリプライマーで作ったパーツも同様にdigestion==
==ラオリプライマーで作ったパーツも同様にdigestion==

Revision as of 13:28, 27 September 2010

Notebook

Digestion of pUC119 by EcoR I, Pst I

－ EcoR I Pst I E, P 古いEcoR I
DNA solution 1 uL 1 uL 1 uL 1 uL 1 uL
DW 17 uL 14 uL 14 uL 13 uL 14 uL
10x M buffer 2 uL 2 uL 2 uL 2 uL 2 uL
0.1% BSA － 2 uL 2 uL 2 uL 2 uL
EcoR I － 1 uL － 1 uL 1 uL
Pst I － － 1 uL 1 uL －
Total 20 uL 20 uL 20 uL 20 uL 20 uL

→Incubated at 37C for 60 min →Electrophoresed 2 uL for confirmation

  • There were no bands, forgot to add DNA :(
  • Reused the remaining 18 uL of digestion solution by adding 1 uL of ADW and 1uL of DNA

→Incubated at 37C for 60 min
→Electrophoresed 2 uL of each solution（+ 0.4 uL 6x SB）

Electrophoresis

Electrophoresis of digestion products
Lane DNA
2 Lambda/HindIII,　EcoR I（4 uL used）
3 Undigested
4 EcoR I
5 Pst I
6 EcoR I + Pst I
7 EcoR I (used old enzyme to check it's activity)
  • In lane 3 monomers, dimers and trimers of plasmid were visible ．
  • From lanes 4 through 7 it's visible that DNA digestion wasn't satisfactory
    • Because plasmid became linear it's was slower than super-coiled one's

ラオリプライマーで作ったパーツも同様にdigestion

　 RBS dT GFP
DNA 1 uL 1 uL 1 uL 1 uL 1 uL 1 uL 1 uL 1 uL 1 uL 1 uL 1 uL 1 uL
DW 17 uL 14 uL 14 uL 13 uL 17 uL 14 uL 14 uL 13 uL 17 uL 14 uL 14 uL 13 uL
10x M buffer 2 uL 2 uL 2 uL 2 uL 2 uL 2 uL 2 uL 2 uL 2 uL 2 uL 2 uL 2 uL
0.1% BSA － 2 uL 2 uL 2 uL － 2 uL 2 uL 2 uL － 2 uL 2 uL 2 uL
EcoR I － 1 uL － 1 uL － 1 uL － 1 uL － 1 uL － 1 uL
Pst I － － 1 uL 1 uL － － 1 uL 1 uL － － 1 uL 1 uL
Total 20 uL 20 uL 20 uL 20 uL 20 uL 20 uL 20 uL 20 uL 20 uL 20 uL 20 uL 20 uL
HokkaidoU Japan 20100830a.jpg

→同様にインキュベート＆電気泳動

電気泳動

  • マーカーはλ/Hind III, EcoR Iと50bp のラダーマーカー
  • ほかは上の表の通り
  • ちゃんと切れてる
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