# Team:HokkaidoU Japan/Notebook/August30

(Difference between revisions)
 Revision as of 12:41, 18 September 2010 (view source)Sprkata (Talk | contribs)← Older edit Latest revision as of 07:59, 27 October 2010 (view source)Sho N (Talk | contribs) (22 intermediate revisions not shown) Line 1: Line 1: - {{Template:HokkaidoU_Japan}} + {{Template:HokkaidoU_Japan}} - =pUC119のEcoR I, Pst I digestion= + - [[Image:HokkaidoU Japan 20100830a.jpg‎|200px|right|thumb|]] + =Digestion of pUC119 by EcoR I, Pst I= - {|width="500px" style="text-align:center;" + + {|border="1" style="text-align:center;" class="protocol" |- |- - |style="border-bottom:1px solid #000;"| + || - |style="border-bottom:1px solid #000;"|－ + |style="border-bottom:1px;"|－ - |style="border-bottom:1px solid #000;"|EcoR I + |style="border-bottom:1px;"|EcoR I - |style="border-bottom:1px solid #000;"|Pst I + |style="border-bottom:1px;"|Pst I - |style="border-bottom:1px solid #000;"|EcoR I, Pst I + |style="border-bottom:1px;"|E, P - |style="border-bottom:1px solid #000;"|古いEcoR I + |style="border-bottom:1px;"|Old EcoR I |- |- - |style="border-right:1px solid #000;"|DNA solution + |DNA solution |1 uL |1 uL |1 uL |1 uL Line 18: Line 19: |1 uL |1 uL |- |- - |style="border-right:1px solid #000;"|DW + |DW |17 uL |17 uL |14 uL |14 uL Line 25: Line 26: |14 uL |14 uL |- |- - |style="border-right:1px solid #000;"|10x M buffer + |10x M buffer |2 uL |2 uL |2 uL |2 uL Line 32: Line 33: |2 uL |2 uL |- |- - |style="border-right:1px solid #000;"|0.1% BSA + |0.1% BSA |－ |－ |2 uL |2 uL Line 39: Line 40: |2 uL |2 uL |- |- - |style="border-right:1px solid #000;"|EcoR I + |EcoR I |－ |－ |1 uL |1 uL Line 46: Line 47: |''1 uL'' |''1 uL'' |- |- - |style="border-right:1px solid #000;"|Pst I + |Pst I |－ |－ |－ |－ Line 53: Line 54: |－ |－ |- |- - |style="border-top:1px solid #000;border-right:1px solid #000;"|'''Total''' + |style="border-top:1px;border-right:1px;"|'''Total''' - |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''' + ||'''20 uL''' - |style="border-top:1px solid #000;"|'''20 uL''' + ||'''20 uL''' |} |} - →37℃, 60 min
+ - →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
+ * 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
- ===電気泳動=== + →Electrophoresed 2 uL of each solution（+ 0.4 uL 6x SB） - {| + - |- + ===Electrophoresis=== - |レーン + - | + [[Image:HokkaidoU Japan 20100830b.jpg‎|200px|right|thumb|Electrophoresis of digestion products]] + + {| class="protocol" + |'''Lane''' + |'''DNA''' |- |- |2 |2 - |λ/''Hin''d III, EcoR I（4 uL使用） + |[http://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 ． - * 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 - ** 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 - + ==Digestion of parts PCRed using digestion visualization primers== - ==ラオリプライマーで作ったパーツも同様にdigestion== + {|style="text-align:center;" class="protocol" - {|width="700px" style="text-align:center; " + |- |- - |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 Line 117: Line 121: |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 Line 131: Line 135: |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 Line 145: Line 149: |2 uL |2 uL |- |- - |style="border-right:1px solid #000;"|0.1% BSA + |style="border-right:1px solid #996;"|0.1% BSA - |0 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 - |0 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 - |0 uL + |－ |2 uL |2 uL |2 uL |2 uL |2 uL |2 uL |- |- - |style="border-right:1px solid #000;"|EcoR I + |style="border-right:1px solid #996;"|EcoR I - |0 uL + |－ |1 uL |1 uL - |0 uL + |－ - |style="border-right:1px solid #000;"|1 uL + |style="border-right:1px solid #996;"|1 uL - |0 uL + |－ |1 uL |1 uL - |0 uL + |－ - |style="border-right:1px solid #000;"|1 uL + |style="border-right:1px solid #996;"|1 uL - |0 uL + |－ |1 uL |1 uL - |0 uL + |－ |1 uL |1 uL |- |- - |style="border-right:1px solid #000;"|Pst I + |style="border-right:1px solid #996;"|Pst I - |0 uL + |－ - |0 uL + |－ |1 uL |1 uL - |style="border-right:1px solid #000;"|1 uL + |style="border-right:1px solid #996;"|1 uL - |0 uL + |－ - |0 uL + |－ |1 uL |1 uL - |style="border-right:1px solid #000;"|1 uL + |style="border-right:1px solid #996;"|1 uL - |0 uL + |－ - |0 uL + |－ |1 uL |1 uL |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 20100830b.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=== - * レーン２のマーカーはλ/''Hin''d III, EcoR I． + * Markers used [http://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''dIII,　EcoR I] and 50bp ladder - * レーン３のマーカーは50bp のラダーマーカー + * Was obvious that parts were cut as intended - * レーン５からは上の表の通り + - * ちゃんと切れてる +

# 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