Team:HokkaidoU Japan/Notebook/August30

From 2010.igem.org

(Difference between revisions)
(ラオリプライマーで作ったパーツも同様にdigestion)
(Digestion of pUC119 by EcoR I, Pst I)
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|style="border-top:1px solid #996;"|20 uL
|style="border-top:1px solid #996;"|20 uL
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[[Image:HokkaidoU Japan 20100830a.jpg‎|200px|right|thumb|]]
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[[Image:HokkaidoU Japan 20100830a.jpg‎|200px|right|thumb|Electrophoresis of digestion products]]
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→同様にインキュベート&電気泳動
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→Incubated at 37C for 60 min
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===電気泳動===
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===Electrophoresis===
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* マーカーはλ/''Hin''d III, EcoR Iと50bp のラダーマーカー
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* Markers used [http://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png Lambda/''Hin''dIII, EcoR I] and 50bp ladder
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* ほかは上の表の通り
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* Was obvious that parts were cut as intended
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* ちゃんと切れてる
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Revision as of 17:05, 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

Digestion of parts PCRed using digestion visualization primers

  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
Electrophoresis of digestion products

→Incubated at 37C for 60 min

Electrophoresis