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Team:HokkaidoU Japan/Notebook/August30
From 2010.igem.org
(Difference between revisions)
(→電気泳動) |
(→ラオリプライマーで作ったパーツも同様にdigestion) |
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| Line 95: | Line 95: | ||
{|style="text-align:center;" class="protocol" | {|style="text-align:center;" class="protocol" | ||
|- | |- | ||
| - | |style="border-right:1px; border-bottom:1px;"| | + | |style="border-right:1px solid #000; border-bottom:1px solid #000;"| |
| - | |colspan="4"|RBS | + | |colspan="4" style="border-right:1px solid #000; border-bottom:1px solid #000;"|RBS |
| - | |colspan="4"|dT | + | |colspan="4" style="border-right:1px solid #000; border-bottom:1px solid #000;"|dT |
| - | |colspan="4"|GFP | + | |colspan="4" style="border-bottom:1px solid #000;"|GFP |
|- | |- | ||
| - | |DNA | + | |style="border-right:1px solid #000;"|DNA |
| - | + | ||
| - | + | ||
|1 uL | |1 uL | ||
|1 uL | |1 uL | ||
|1 uL | |1 uL | ||
| + | |style="border-right:1px solid #000;"|1 uL | ||
|1 uL | |1 uL | ||
|1 uL | |1 uL | ||
|1 uL | |1 uL | ||
| + | |style="border-right:1px solid #000;"|1 uL | ||
|1 uL | |1 uL | ||
|1 uL | |1 uL | ||
| Line 114: | Line 114: | ||
|1 uL | |1 uL | ||
|- | |- | ||
| - | |DW | + | |style="border-right:1px solid #000;"|DW |
|17 uL | |17 uL | ||
|14 uL | |14 uL | ||
|14 uL | |14 uL | ||
| - | |13 uL | + | |style="border-right:1px solid #000;"|13 uL |
|17 uL | |17 uL | ||
|14 uL | |14 uL | ||
|14 uL | |14 uL | ||
| - | |13 uL | + | |style="border-right:1px solid #000;"|13 uL |
|17 uL | |17 uL | ||
|14 uL | |14 uL | ||
| Line 128: | Line 128: | ||
|13 uL | |13 uL | ||
|- | |- | ||
| - | |10x M buffer | + | |style="border-right:1px solid #000;"|10x M buffer |
| - | + | ||
| - | + | ||
|2 uL | |2 uL | ||
|2 uL | |2 uL | ||
|2 uL | |2 uL | ||
| + | |style="border-right:1px solid #000;"|2 uL | ||
|2 uL | |2 uL | ||
|2 uL | |2 uL | ||
|2 uL | |2 uL | ||
| + | |style="border-right:1px solid #000;"|2 uL | ||
|2 uL | |2 uL | ||
|2 uL | |2 uL | ||
| Line 142: | Line 142: | ||
|2 uL | |2 uL | ||
|- | |- | ||
| - | |0.1% BSA | + | |style="border-right:1px solid #000;"|0.1% BSA |
|0 uL | |0 uL | ||
|2 uL | |2 uL | ||
|2 uL | |2 uL | ||
| - | |2 uL | + | |style="border-right:1px solid #000;"|2 uL |
|0 uL | |0 uL | ||
|2 uL | |2 uL | ||
|2 uL | |2 uL | ||
| - | |2 uL | + | |style="border-right:1px solid #000;"|2 uL |
|0 uL | |0 uL | ||
|2 uL | |2 uL | ||
| Line 156: | Line 156: | ||
|2 uL | |2 uL | ||
|- | |- | ||
| - | |EcoR I | + | |style="border-right:1px solid #000;"|EcoR I |
|0 uL | |0 uL | ||
|1 uL | |1 uL | ||
|0 uL | |0 uL | ||
| - | |1 uL | + | |style="border-right:1px solid #000;"|1 uL |
|0 uL | |0 uL | ||
|1 uL | |1 uL | ||
|0 uL | |0 uL | ||
| - | |1 uL | + | |style="border-right:1px solid #000;"|1 uL |
|0 uL | |0 uL | ||
|1 uL | |1 uL | ||
| Line 170: | Line 170: | ||
|1 uL | |1 uL | ||
|- | |- | ||
| - | |Pst I | + | |style="border-right:1px solid #000;"|Pst I |
|0 uL | |0 uL | ||
|0 uL | |0 uL | ||
|1 uL | |1 uL | ||
| - | |1 uL | + | |style="border-right:1px solid #000;"|1 uL |
|0 uL | |0 uL | ||
|0 uL | |0 uL | ||
|1 uL | |1 uL | ||
| - | |1 uL | + | |style="border-right:1px solid #000;"|1 uL |
|0 uL | |0 uL | ||
|0 uL | |0 uL | ||
| Line 184: | Line 184: | ||
|1 uL | |1 uL | ||
|- | |- | ||
| - | ||Total | + | |style="border-right:1px solid #000; border-top:1px solid #000;"|Total |
| - | ||20 uL | + | |style="border-top:1px solid #000;"|20 uL |
| - | ||20 uL | + | |style="border-top:1px solid #000;"|20 uL |
| - | ||20 uL | + | |style="border-top:1px solid #000;"|20 uL |
| - | |20 uL | + | |style="border-top:1px solid #000; border-right:1px solid #000;"|20 uL |
| - | ||20 uL | + | |style="border-top:1px solid #000;"|20 uL |
| - | ||20 uL | + | |style="border-top:1px solid #000;"|20 uL |
| - | ||20 uL | + | |style="border-top:1px solid #000;"|20 uL |
| - | |20 uL | + | |style="border-top:1px solid #000; border-right:1px solid #000;"|20 uL |
| - | ||20 uL | + | |style="border-top:1px solid #000;"|20 uL |
| - | ||20 uL | + | |style="border-top:1px solid #000;"|20 uL |
| - | ||20 uL | + | |style="border-top:1px solid #000;"|20 uL |
| - | ||20 uL | + | |style="border-top:1px solid #000;"|20 uL |
|} | |} | ||
[[Image:HokkaidoU Japan 20100830a.jpg|200px|right|thumb|]] | [[Image:HokkaidoU Japan 20100830a.jpg|200px|right|thumb|]] | ||
Revision as of 05:50, 19 September 2010
since 13 Jul 2010
since 1 Aug 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
| 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
