Team:HokkaidoU Japan/Notebook/August30
From 2010.igem.org
(Difference between revisions)
(→pUC119のEcoR I, Pst I digestion) |
(→pUC119のEcoR I, Pst I digestion) |
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- | = | + | =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 65: | Line 65: | ||
→Electrophoresed 2 uL for confirmation | →Electrophoresed 2 uL for confirmation | ||
* There were no bands, forgot to add DNA :( | * 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<br> | |
- | + | →Electrophoresed 2 uL of each solution(+ 0.4 uL 6x SB) | |
===Electrophoresis=== | ===Electrophoresis=== | ||
- | [[Image:HokkaidoU Japan 20100830b.jpg|200px|right|thumb|]] | + | [[Image:HokkaidoU Japan 20100830b.jpg|200px|right|thumb|Electrophoresis of digestion products]] |
+ | |||
{| class="protocol" | {| class="protocol" | ||
|'''Lane''' | |'''Lane''' | ||
Line 77: | Line 78: | ||
|- | |- | ||
|2 | |2 | ||
- | | | + | |[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png Lambda/''Hin''dIII, EcoR I](4 uL used) |
|- | |- | ||
|3 | |3 | ||
- | | | + | |Undigested |
|- | |- | ||
|4 | |4 | ||
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|- | |- | ||
|7 | |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 |
==ラオリプライマーで作ったパーツも同様にdigestion== | ==ラオリプライマーで作ったパーツも同様にdigestion== |
Revision as of 13:28, 27 September 2010
Digestion of pUC119 by EcoR I, Pst I
- | 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 |
→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
Lane | DNA |
2 | Lambda/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
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 | - | 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 |
→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