Team:HokkaidoU Japan/Notebook/August30

From 2010.igem.org

(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]]
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{| 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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|[http://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

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