Team:HokkaidoU Japan/Notebook/August23
From 2010.igem.org
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+ | =pSB1C3の活性評価= | ||
+ | ==EcoR I/Pst I Digestion== | ||
+ | ===反応系=== | ||
+ | {|style="text-align:center;" | ||
+ | |- | ||
+ | |pSB1C3 | ||
+ | |50 uL | ||
+ | |- | ||
+ | |DW | ||
+ | |4 uL | ||
+ | |- | ||
+ | |0.1% BSA | ||
+ | |7 uL | ||
+ | |- | ||
+ | |10x M Buffer | ||
+ | |7 uL | ||
+ | |- | ||
+ | |EcoR I | ||
+ | |1 uL | ||
+ | |- | ||
+ | |Pst I | ||
+ | |1 uL | ||
+ | |- | ||
+ | |style="border-top:1px solid #000;"|'''Total''' | ||
+ | |style="border-top:1px solid #000;"|'''70 uL''' | ||
+ | |} | ||
+ | |||
+ | * 37℃,60 min | ||
+ | * 15 uL 6x Sample Bufferを加え,6レーンに分けて泳動 | ||
+ | * ゲル抽出 | ||
+ | |||
+ | ===Ligationあり=== | ||
+ | ゲル抽して得た100 uL DNAのうち2 uLを使用してTransformation | ||
+ | |||
+ | ===Ligationなし=== | ||
+ | ゲル抽して得た100 uL DNAのうち2 uLに2 uL Ligation Solutionを加える<br> | ||
+ | 16℃で30分おき,計4 uLをTransformation | ||
+ | |||
+ | =VectorのPCRリベンジ= | ||
+ | [[Image:HokkaidoU Japan 20100823a.JPG|200px|right|thumb|]] | ||
+ | pSB1A3, pSB1C3, pSB1T3それぞれについて以下の反応系でPCRを行った | ||
+ | {|style="text-align:center;" | ||
+ | |- | ||
+ | |Vector | ||
+ | |1 | ||
+ | |- | ||
+ | |DW | ||
+ | |33 | ||
+ | |- | ||
+ | |10x Buffer | ||
+ | |5 | ||
+ | |- | ||
+ | |2 M 4dNTPs | ||
+ | |5 | ||
+ | |- | ||
+ | |25 mM MgSO<sub>4</sub> | ||
+ | |3 | ||
+ | |- | ||
+ | |Suffix-F | ||
+ | |1 | ||
+ | |- | ||
+ | |Prefix-R | ||
+ | |1 | ||
+ | |- | ||
+ | |KOD | ||
+ | |1 | ||
+ | |- | ||
+ | |style="border-top:1px solid #000;"|'''Total''' | ||
+ | |style="border-top:1px solid #000;"|'''50 uL''' | ||
+ | |} | ||
+ | |||
+ | * 前回と違い,Extentionを120 secで行った |
Revision as of 11:58, 18 September 2010
pSB1C3の活性評価
EcoR I/Pst I Digestion
反応系
pSB1C3 | 50 uL |
DW | 4 uL |
0.1% BSA | 7 uL |
10x M Buffer | 7 uL |
EcoR I | 1 uL |
Pst I | 1 uL |
Total | 70 uL |
- 37℃,60 min
- 15 uL 6x Sample Bufferを加え,6レーンに分けて泳動
- ゲル抽出
Ligationあり
ゲル抽して得た100 uL DNAのうち2 uLを使用してTransformation
Ligationなし
ゲル抽して得た100 uL DNAのうち2 uLに2 uL Ligation Solutionを加える
16℃で30分おき,計4 uLをTransformation
VectorのPCRリベンジ
pSB1A3, pSB1C3, pSB1T3それぞれについて以下の反応系でPCRを行った
Vector | 1 |
DW | 33 |
10x Buffer | 5 |
2 M 4dNTPs | 5 |
25 mM MgSO4 | 3 |
Suffix-F | 1 |
Prefix-R | 1 |
KOD | 1 |
Total | 50 uL |
- 前回と違い,Extentionを120 secで行った