Team:HokkaidoU Japan/Notebook/August30
From 2010.igem.org
(Difference between revisions)
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=pUC119のEcoR I, Pst I digestion= | =pUC119のEcoR I, Pst I digestion= | ||
- | {|style="text-align:center;" class="protocol" | + | {|border="1px"style="text-align:center;" class="protocol" |
|- | |- | ||
- | | | + | || |
|style="border-bottom:1px;"|- | |style="border-bottom:1px;"|- | ||
|style="border-bottom:1px;"|EcoR I | |style="border-bottom:1px;"|EcoR I | ||
|style="border-bottom:1px;"|Pst I | |style="border-bottom:1px;"|Pst I | ||
- | |style="border-bottom:1px;"| | + | |style="border-bottom:1px;"|E, P |
|style="border-bottom:1px;"|古いEcoR I | |style="border-bottom:1px;"|古いEcoR I | ||
|- | |- | ||
- | + | |DNA solution | |
|1 uL | |1 uL | ||
|1 uL | |1 uL | ||
Line 18: | Line 18: | ||
|1 uL | |1 uL | ||
|- | |- | ||
- | + | |DW | |
|17 uL | |17 uL | ||
|14 uL | |14 uL | ||
Line 25: | Line 25: | ||
|14 uL | |14 uL | ||
|- | |- | ||
- | + | |10x M buffer | |
|2 uL | |2 uL | ||
|2 uL | |2 uL | ||
Line 32: | Line 32: | ||
|2 uL | |2 uL | ||
|- | |- | ||
- | + | |0.1% BSA | |
|- | |- | ||
|2 uL | |2 uL | ||
Line 39: | Line 39: | ||
|2 uL | |2 uL | ||
|- | |- | ||
- | + | |EcoR I | |
|- | |- | ||
|1 uL | |1 uL | ||
Line 46: | Line 46: | ||
|''1 uL'' | |''1 uL'' | ||
|- | |- | ||
- | + | |Pst I | |
|- | |- | ||
|- | |- | ||
Line 54: | Line 54: | ||
|- | |- | ||
|style="border-top:1px;border-right:1px;"|'''Total''' | |style="border-top:1px;border-right:1px;"|'''Total''' | ||
- | | | + | ||'''20 uL''' |
- | | | + | ||'''20 uL''' |
- | | | + | ||'''20 uL''' |
- | | | + | ||'''20 uL''' |
- | | | + | ||'''20 uL''' |
|} | |} | ||
→37℃, 60 min<br> | →37℃, 60 min<br> | ||
Line 100: | Line 100: | ||
|- | |- | ||
|style="border-right:1px; border-bottom:1px;"| | |style="border-right:1px; border-bottom:1px;"| | ||
- | |colspan="4 | + | |colspan="4"|RBS |
- | |colspan="4 | + | |colspan="4"|dT |
- | |colspan="4 | + | |colspan="4"|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 | ||
|1 uL | |1 uL | ||
- | |||
|1 uL | |1 uL | ||
|1 uL | |1 uL | ||
Line 118: | Line 118: | ||
|1 uL | |1 uL | ||
|- | |- | ||
- | + | |DW | |
|17 uL | |17 uL | ||
|14 uL | |14 uL | ||
|14 uL | |14 uL | ||
- | + | |13 uL | |
|17 uL | |17 uL | ||
|14 uL | |14 uL | ||
|14 uL | |14 uL | ||
- | + | |13 uL | |
|17 uL | |17 uL | ||
|14 uL | |14 uL | ||
Line 132: | Line 132: | ||
|13 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 | ||
|2 uL | |2 uL | ||
- | |||
|2 uL | |2 uL | ||
|2 uL | |2 uL | ||
Line 146: | Line 146: | ||
|2 uL | |2 uL | ||
|- | |- | ||
- | + | |0.1% BSA | |
|0 uL | |0 uL | ||
|2 uL | |2 uL | ||
|2 uL | |2 uL | ||
- | + | |2 uL | |
|0 uL | |0 uL | ||
|2 uL | |2 uL | ||
|2 uL | |2 uL | ||
- | + | |2 uL | |
|0 uL | |0 uL | ||
|2 uL | |2 uL | ||
Line 160: | Line 160: | ||
|2 uL | |2 uL | ||
|- | |- | ||
- | + | |EcoR I | |
|0 uL | |0 uL | ||
|1 uL | |1 uL | ||
|0 uL | |0 uL | ||
- | + | |1 uL | |
|0 uL | |0 uL | ||
|1 uL | |1 uL | ||
|0 uL | |0 uL | ||
- | + | |1 uL | |
|0 uL | |0 uL | ||
|1 uL | |1 uL | ||
Line 174: | Line 174: | ||
|1 uL | |1 uL | ||
|- | |- | ||
- | + | |Pst I | |
|0 uL | |0 uL | ||
|0 uL | |0 uL | ||
|1 uL | |1 uL | ||
- | + | |1 uL | |
|0 uL | |0 uL | ||
|0 uL | |0 uL | ||
|1 uL | |1 uL | ||
- | + | |1 uL | |
|0 uL | |0 uL | ||
|0 uL | |0 uL | ||
Line 188: | Line 188: | ||
|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 |
|} | |} | ||
[[Image:HokkaidoU Japan 20100830a.jpg|200px|right|thumb|]] | [[Image:HokkaidoU Japan 20100830a.jpg|200px|right|thumb|]] |
Revision as of 05:47, 19 September 2010
pUC119のEcoR I, Pst I digestion
- | 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 |
→37℃, 60 min
→2 uLを電気泳動で確認
- バカ橋さんがDNAを入れ忘れたためやりなおし
- 残った18 uLにDW1 uLとDNAを加えて
→37℃, 60 min
→2 uLを電気泳動で確認(+ 0.4 uL 6x SB)
電気泳動
レーン | |
2 | λ/Hind III, EcoR I(4 uL使用) |
3 | 切ってない |
4 | EcoR I |
5 | Pst I |
6 | EcoR I + Pst I |
7 | 古いEcoR I |
- 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.
- レーン4~7ではこれらが切れているのが分かる
- 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
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 | 0 uL | 2 uL | 2 uL | 2 uL | 0 uL | 2 uL | 2 uL | 2 uL | 0 uL | 2 uL | 2 uL | 2 uL |
EcoR I | 0 uL | 1 uL | 0 uL | 1 uL | 0 uL | 1 uL | 0 uL | 1 uL | 0 uL | 1 uL | 0 uL | 1 uL |
Pst I | 0 uL | 0 uL | 1 uL | 1 uL | 0 uL | 0 uL | 1 uL | 1 uL | 0 uL | 0 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 |
→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