Team:HokkaidoU Japan/Notebook/August30

From 2010.igem.org

(Difference between revisions)
(pUC119のEcoR I, Pst I digestion)
 
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|style="border-bottom:1px;"|Pst I
|style="border-bottom:1px;"|Pst I
|style="border-bottom:1px;"|E, P
|style="border-bottom:1px;"|E, P
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|style="border-bottom:1px;"|古いEcoR I
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|style="border-bottom:1px;"|Old EcoR I
|-
|-
|DNA solution
|DNA solution
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→Incubated at 37C for 60 min
→Incubated at 37C for 60 min
→Electrophoresed 2 uL for confirmation
→Electrophoresed 2 uL for confirmation
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* There were no bands, forgot to add DNA :(
+
* 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
* Reused the remaining 18 uL of digestion solution by adding 1 uL of ADW and 1uL of DNA
→Incubated at 37C for 60 min<br>
→Incubated at 37C for 60 min<br>
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|-
|-
|2
|2
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|[http://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png Lambda/''Hin''dIII, EcoR I](4 uL used)
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|[http://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''dIII, EcoR I](4 uL used)
|-
|-
|3
|3
Line 99: Line 99:
** Because plasmid became linear it's was slower than super-coiled one's
** Because plasmid became linear it's was slower than super-coiled one's
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==ラオリプライマーで作ったパーツも同様にdigestion==
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==Digestion of parts PCRed using digestion visualization primers==
{|style="text-align:center;" class="protocol"
{|style="text-align:center;" class="protocol"
|-
|-
|style="border-right:1px solid #996; border-bottom:1px solid #996;"| 
|style="border-right:1px solid #996; border-bottom:1px solid #996;"| 
|colspan="4" style="border-right:1px solid #996; border-bottom:1px solid #996;"|RBS
|colspan="4" style="border-right:1px solid #996; border-bottom:1px solid #996;"|RBS
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|colspan="4" style="border-right:1px solid #996; border-bottom:1px solid #996;"|dT
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|colspan="4" style="border-right:1px solid #996; border-bottom:1px solid #996;"|double terminator
|colspan="4" style="border-bottom:1px solid #996;"|GFP
|colspan="4" style="border-bottom:1px solid #996;"|GFP
|-
|-
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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 λ/''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
+

Latest revision as of 07:59, 27 October 2010

Digestion of pUC119 by EcoR I, Pst I

EcoR I Pst I E, P Old 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 λ/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 double terminator 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