Team:HokkaidoU Japan/Notebook/August30

From 2010.igem.org

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(pUC119のEcoR I, Pst I digestion)
 
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=pUC119のEcoR I, Pst I digestion=
+
=Digestion of pUC119 by EcoR I, Pst I=
-
{|style="text-align:center;" class="protocol"
+
 
 +
{|border="1" style="text-align:center;" class="protocol"
|-
|-
-
|style="border-bottom:1px solid #000;border-right:1px solid #000;"|
+
||
-
|style="border-bottom:1px solid #000;"|-
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|style="border-bottom:1px;"|-
-
|style="border-bottom:1px solid #000;"|EcoR I
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|style="border-bottom:1px;"|EcoR I
-
|style="border-bottom:1px solid #000;"|Pst I
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|style="border-bottom:1px;"|Pst I
-
|style="border-bottom:1px solid #000;"|EcoR I, Pst I
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|style="border-bottom:1px;"|E, P
-
|style="border-bottom:1px solid #000;"|古いEcoR I
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|style="border-bottom:1px;"|Old EcoR I
|-
|-
-
|style="border-right:1px solid #000;"|DNA solution
+
|DNA solution
|1 uL
|1 uL
|1 uL
|1 uL
Line 18: Line 19:
|1 uL
|1 uL
|-
|-
-
|style="border-right:1px solid #000;"|DW
+
|DW
|17 uL
|17 uL
|14 uL
|14 uL
Line 25: Line 26:
|14 uL
|14 uL
|-
|-
-
|style="border-right:1px solid #000;"|10x M buffer
+
|10x M buffer
|2 uL
|2 uL
|2 uL
|2 uL
Line 32: Line 33:
|2 uL
|2 uL
|-
|-
-
|style="border-right:1px solid #000;"|0.1% BSA
+
|0.1% BSA
|-
|-
|2 uL
|2 uL
Line 39: Line 40:
|2 uL
|2 uL
|-
|-
-
|style="border-right:1px solid #000;"|EcoR I
+
|EcoR I
|-
|-
|1 uL
|1 uL
Line 46: Line 47:
|''1 uL''
|''1 uL''
|-
|-
-
|style="border-right:1px solid #000;"|Pst I
+
|Pst I
|-
|-
|-
|-
Line 53: Line 54:
|-
|-
|-
|-
-
|style="border-top:1px solid #000;border-right:1px solid #000;"|'''Total'''
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|style="border-top:1px;border-right:1px;"|'''Total'''
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|style="border-top:1px solid #000;"|'''20 uL'''
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||'''20 uL'''
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|style="border-top:1px solid #000;"|'''20 uL'''
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||'''20 uL'''
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|style="border-top:1px solid #000;"|'''20 uL'''
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||'''20 uL'''
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|style="border-top:1px solid #000;"|'''20 uL'''
+
||'''20 uL'''
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|style="border-top:1px solid #000;"|'''20 uL'''
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||'''20 uL'''
|}
|}
-
→37℃, 60 min<br>
+
 
-
→2 uLを電気泳動で確認
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→Incubated at 37C for 60 min
-
* バカ橋さんがDNAを入れ忘れたためやりなおし
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→Electrophoresed 2 uL for confirmation
-
* 残った18 uLにDW1 uLとDNAを加えて
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* There were no bands, forgot to add DNA
-
→37℃, 60 min<br>
+
* Reused the remaining 18 uL of digestion solution by adding 1 uL of ADW and 1uL of DNA
-
→2 uLを電気泳動で確認(+ 0.4 uL 6x SB)
+
→Incubated at 37C for 60 min<br>
-
===電気泳動===
+
→Electrophoresed 2 uL of each solution(+ 0.4 uL 6x SB)
-
[[Image:HokkaidoU Japan 20100830b.jpg‎|200px|right|thumb|]]
+
 
 +
===Electrophoresis===
 +
 
 +
[[Image:HokkaidoU Japan 20100830b.jpg‎|200px|right|thumb|Electrophoresis of digestion products]]
 +
 
{| class="protocol"
{| class="protocol"
-
|-
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|'''Lane'''
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|レーン
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|'''DNA'''
-
|
+
|-
|-
|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 λ/''Hin''dIII, EcoR I](4 uL used)
|-
|-
|3
|3
-
|切ってない
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|Undigested
|-
|-
|4
|4
Line 89: Line 93:
|-
|-
|7
|7
-
|古いEcoR I
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|EcoR I (used old enzyme to check it's activity)
|}
|}
-
* 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.
+
* 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
-
 
+
==Digestion of parts PCRed using digestion visualization primers==
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==ラオリプライマーで作ったパーツも同様にdigestion==
+
{|style="text-align:center;" class="protocol"
{|style="text-align:center;" class="protocol"
|-
|-
-
|style="border-right:1px solid #000; border-bottom:1px solid #000;"| 
+
|style="border-right:1px solid #996; border-bottom:1px solid #996;"| 
-
|colspan="4" style="border-right:1px solid #000; border-bottom:1px solid #000;"|RBS
+
|colspan="4" style="border-right:1px solid #996; border-bottom:1px solid #996;"|RBS
-
|colspan="4" style="border-right:1px solid #000; border-bottom:1px solid #000;"|dT
+
|colspan="4" style="border-right:1px solid #996; border-bottom:1px solid #996;"|double terminator
-
|colspan="4" style="border-bottom:1px solid #000;"|GFP
+
|colspan="4" style="border-bottom:1px solid #996;"|GFP
|-
|-
-
|style="border-right:1px solid #000;"|DNA
+
|style="border-right:1px solid #996;"|DNA
|1 uL
|1 uL
|1 uL
|1 uL
|1 uL
|1 uL
-
|style="border-right:1px solid #000;"|1 uL
+
|style="border-right:1px solid #996;"|1 uL
|1 uL
|1 uL
|1 uL
|1 uL
|1 uL
|1 uL
-
|style="border-right:1px solid #000;"|1 uL
+
|style="border-right:1px solid #996;"|1 uL
|1 uL
|1 uL
|1 uL
|1 uL
Line 118: Line 121:
|1 uL
|1 uL
|-
|-
-
|style="border-right:1px solid #000;"|DW
+
|style="border-right:1px solid #996;"|DW
|17 uL
|17 uL
|14 uL
|14 uL
|14 uL
|14 uL
-
|style="border-right:1px solid #000;"|13 uL
+
|style="border-right:1px solid #996;"|13 uL
|17 uL
|17 uL
|14 uL
|14 uL
|14 uL
|14 uL
-
|style="border-right:1px solid #000;"|13 uL
+
|style="border-right:1px solid #996;"|13 uL
|17 uL
|17 uL
|14 uL
|14 uL
Line 132: Line 135:
|13 uL
|13 uL
|-
|-
-
|style="border-right:1px solid #000;"|10x M buffer
+
|style="border-right:1px solid #996;"|10x M buffer
|2 uL
|2 uL
|2 uL
|2 uL
|2 uL
|2 uL
-
|style="border-right:1px solid #000;"|2 uL
+
|style="border-right:1px solid #996;"|2 uL
|2 uL
|2 uL
|2 uL
|2 uL
|2 uL
|2 uL
-
|style="border-right:1px solid #000;"|2 uL
+
|style="border-right:1px solid #996;"|2 uL
|2 uL
|2 uL
|2 uL
|2 uL
Line 146: Line 149:
|2 uL
|2 uL
|-
|-
-
|style="border-right:1px solid #000;"|0.1% BSA
+
|style="border-right:1px solid #996;"|0.1% BSA
-
|0 uL
+
|
|2 uL
|2 uL
|2 uL
|2 uL
-
|style="border-right:1px solid #000;"|2 uL
+
|style="border-right:1px solid #996;"|2 uL
-
|0 uL
+
|
|2 uL
|2 uL
|2 uL
|2 uL
-
|style="border-right:1px solid #000;"|2 uL
+
|style="border-right:1px solid #996;"|2 uL
-
|0 uL
+
|
|2 uL
|2 uL
|2 uL
|2 uL
|2 uL
|2 uL
|-
|-
-
|style="border-right:1px solid #000;"|EcoR I
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|style="border-right:1px solid #996;"|EcoR I
-
|0 uL
+
|
|1 uL
|1 uL
-
|0 uL
+
|
-
|style="border-right:1px solid #000;"|1 uL
+
|style="border-right:1px solid #996;"|1 uL
-
|0 uL
+
|
|1 uL
|1 uL
-
|0 uL
+
|
-
|style="border-right:1px solid #000;"|1 uL
+
|style="border-right:1px solid #996;"|1 uL
-
|0 uL
+
|
|1 uL
|1 uL
-
|0 uL
+
|
|1 uL
|1 uL
|-
|-
-
|style="border-right:1px solid #000;"|Pst I
+
|style="border-right:1px solid #996;"|Pst I
-
|0 uL
+
|
-
|0 uL
+
|
|1 uL
|1 uL
-
|style="border-right:1px solid #000;"|1 uL
+
|style="border-right:1px solid #996;"|1 uL
-
|0 uL
+
|
-
|0 uL
+
|
|1 uL
|1 uL
-
|style="border-right:1px solid #000;"|1 uL
+
|style="border-right:1px solid #996;"|1 uL
-
|0 uL
+
|
-
|0 uL
+
|
|1 uL
|1 uL
|1 uL
|1 uL
|-
|-
-
|style="border-right:1px solid #000; border-top:1px solid #000;"|Total
+
|style="border-right:1px solid #996; border-top:1px solid #996;"|Total
-
|style="border-top:1px solid #000;"|20 uL
+
|style="border-top:1px solid #996;"|20 uL
-
|style="border-top:1px solid #000;"|20 uL
+
|style="border-top:1px solid #996;"|20 uL
-
|style="border-top:1px solid #000;"|20 uL
+
|style="border-top:1px solid #996;"|20 uL
-
|style="border-top:1px solid #000; border-right:1px solid #000;"|20 uL
+
|style="border-top:1px solid #996; border-right:1px solid #996;"|20 uL
-
|style="border-top:1px solid #000;"|20 uL
+
|style="border-top:1px solid #996;"|20 uL
-
|style="border-top:1px solid #000;"|20 uL
+
|style="border-top:1px solid #996;"|20 uL
-
|style="border-top:1px solid #000;"|20 uL
+
|style="border-top:1px solid #996;"|20 uL
-
|style="border-top:1px solid #000; border-right:1px solid #000;"|20 uL
+
|style="border-top:1px solid #996; border-right:1px solid #996;"|20 uL
-
|style="border-top:1px solid #000;"|20 uL
+
|style="border-top:1px solid #996;"|20 uL
-
|style="border-top:1px solid #000;"|20 uL
+
|style="border-top:1px solid #996;"|20 uL
-
|style="border-top:1px solid #000;"|20 uL
+
|style="border-top:1px solid #996;"|20 uL
-
|style="border-top:1px solid #000;"|20 uL
+
|style="border-top:1px solid #996;"|20 uL
|}
|}
-
[[Image:HokkaidoU Japan 20100830a.jpg‎|200px|right|thumb|]]
+
[[Image:HokkaidoU Japan 20100830a.jpg‎|200px|right|thumb|Electrophoresis of digestion products]]
-
→同様にインキュベート&電気泳動
+
→Incubated at 37C for 60 min
-
===電気泳動===
+
===Electrophoresis===
-
* マーカーはλ/''Hin''d III, EcoR Iと50bp のラダーマーカー
+
* Markers used [https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''dIII, EcoR I] and 50bp ladder
-
* ほかは上の表の通り
+
* Was obvious that parts were cut as intended
-
* 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