Team:HokkaidoU Japan/Notebook/August30

From 2010.igem.org

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=pUC119のEcoR I, Pst I digestion=
+
=Digestion of pUC119 by EcoR I, Pst I=
 +
 
{|border="1" style="text-align:center;" class="protocol"
{|border="1" style="text-align:center;" class="protocol"
|-
|-
Line 9: Line 10:
|style="border-bottom:1px;"|Pst I
|style="border-bottom:1px;"|Pst I
|style="border-bottom:1px;"|E, P
|style="border-bottom:1px;"|E, P
-
|style="border-bottom:1px;"|古いEcoR I
+
|style="border-bottom:1px;"|Old EcoR I
|-
|-
|DNA solution
|DNA solution
Line 60: Line 61:
||'''20 uL'''
||'''20 uL'''
|}
|}
-
→37℃, 60 min<br>
+
 
-
→2 uLを電気泳動で確認
+
→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
+
→Electrophoresed 2 uL for confirmation
-
* 残った18 uLにDW1 uLとDNAを加えて
+
* 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"
|'''Lane'''
|'''Lane'''
Line 73: Line 78:
|-
|-
|2
|2
-
|λ/''Hin''d III, EcoR I（4 uL使用）
+
|[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
+
|Undigested
|-
|-
|4
|4
Line 88: Line 93:
|-
|-
|7
|7
-
|古いEcoR I
+
|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
+
* 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
+
** 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==
+
==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 #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
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|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
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|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
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|2 uL
|2 uL
|-
|-
-
|style="border-right:1px solid #000;"|0.1% BSA
+
|style="border-right:1px solid #996;"|0.1% BSA
|－
|－
|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
-
|style="border-right:1px solid #000;"|2 uL
+
|style="border-right:1px solid #996;"|2 uL
|－
|－
|2 uL
|2 uL
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|2 uL
|2 uL
|-
|-
-
|style="border-right:1px solid #000;"|EcoR I
+
|style="border-right:1px solid #996;"|EcoR I
|－
|－
|1 uL
|1 uL
|－
|－
-
|style="border-right:1px solid #000;"|1 uL
+
|style="border-right:1px solid #996;"|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
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|1 uL
|1 uL
|-
|-
-
|style="border-right:1px solid #000;"|Pst I
+
|style="border-right:1px solid #996;"|Pst I
|－
|－
|－
|－
|1 uL
|1 uL
-
|style="border-right:1px solid #000;"|1 uL
+
|style="border-right:1px solid #996;"|1 uL
|－
|－
|－
|－
|1 uL
|1 uL
-
|style="border-right:1px solid #000;"|1 uL
+
|style="border-right:1px solid #996;"|1 uL
|－
|－
|－
|－
Line 186: Line 191:
|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

Notebook

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

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