Team:HokkaidoU Japan/Notebook/August30

From 2010.igem.org

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{{Template:HokkaidoU_Japan}}<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/August27|August 27]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/August31|August 31]]</div></div>
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=Digestion of pUC119 by EcoR I, Pst I=
 +
 
 +
{|border="1" style="text-align:center;" class="protocol"
 +
|-
 +
||
 +
|style="border-bottom:1px;"|－
 +
|style="border-bottom:1px;"|EcoR I
 +
|style="border-bottom:1px;"|Pst I
 +
|style="border-bottom:1px;"|E, P
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|style="border-bottom:1px;"|Old EcoR I
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|-
 +
|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
 +
|－
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|-
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|style="border-top:1px;border-right:1px;"|'''Total'''
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||'''20 uL'''
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||'''20 uL'''
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||'''20 uL'''
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||'''20 uL'''
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||'''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<br>
 +
→Electrophoresed 2 uL of each solution（+ 0.4 uL 6x SB）
 +
 
 +
===Electrophoresis===
 +
 
 +
[[Image:HokkaidoU Japan 20100830b.jpg‎|200px|right|thumb|Electrophoresis of digestion products]]
 +
 
 +
{| class="protocol"
 +
|'''Lane'''
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|'''DNA'''
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|-
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|2
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|[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''dIII,　EcoR I]（4 uL used）
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|-
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|3
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|Undigested
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|-
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|4
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|EcoR I
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|-
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|5
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|Pst I
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|-
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|6
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|EcoR I + Pst I
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|-
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|7
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|EcoR I (used old enzyme to check it's activity)
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|}
 +
* 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==
 +
{|style="text-align:center;" class="protocol"
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|-
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|style="border-right:1px solid #996; border-bottom:1px solid #996;"|　
 +
|colspan="4" style="border-right:1px solid #996; border-bottom:1px solid #996;"|RBS
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|colspan="4" style="border-right:1px solid #996; border-bottom:1px solid #996;"|double terminator
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|colspan="4" style="border-bottom:1px solid #996;"|GFP
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|-
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|style="border-right:1px solid #996;"|DNA
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|1 uL
 +
|1 uL
 +
|1 uL
 +
|style="border-right:1px solid #996;"|1 uL
 +
|1 uL
 +
|1 uL
 +
|1 uL
 +
|style="border-right:1px solid #996;"|1 uL
 +
|1 uL
 +
|1 uL
 +
|1 uL
 +
|1 uL
 +
|-
 +
|style="border-right:1px solid #996;"|DW
 +
|17 uL
 +
|14 uL
 +
|14 uL
 +
|style="border-right:1px solid #996;"|13 uL
 +
|17 uL
 +
|14 uL
 +
|14 uL
 +
|style="border-right:1px solid #996;"|13 uL
 +
|17 uL
 +
|14 uL
 +
|14 uL
 +
|13 uL
 +
|-
 +
|style="border-right:1px solid #996;"|10x M buffer
 +
|2 uL
 +
|2 uL
 +
|2 uL
 +
|style="border-right:1px solid #996;"|2 uL
 +
|2 uL
 +
|2 uL
 +
|2 uL
 +
|style="border-right:1px solid #996;"|2 uL
 +
|2 uL
 +
|2 uL
 +
|2 uL
 +
|2 uL
 +
|-
 +
|style="border-right:1px solid #996;"|0.1% BSA
 +
|－
 +
|2 uL
 +
|2 uL
 +
|style="border-right:1px solid #996;"|2 uL
 +
|－
 +
|2 uL
 +
|2 uL
 +
|style="border-right:1px solid #996;"|2 uL
 +
|－
 +
|2 uL
 +
|2 uL
 +
|2 uL
 +
|-
 +
|style="border-right:1px solid #996;"|EcoR I
 +
|－
 +
|1 uL
 +
|－
 +
|style="border-right:1px solid #996;"|1 uL
 +
|－
 +
|1 uL
 +
|－
 +
|style="border-right:1px solid #996;"|1 uL
 +
|－
 +
|1 uL
 +
|－
 +
|1 uL
 +
|-
 +
|style="border-right:1px solid #996;"|Pst I
 +
|－
 +
|－
 +
|1 uL
 +
|style="border-right:1px solid #996;"|1 uL
 +
|－
 +
|－
 +
|1 uL
 +
|style="border-right:1px solid #996;"|1 uL
 +
|－
 +
|－
 +
|1 uL
 +
|1 uL
 +
|-
 +
|style="border-right:1px solid #996; border-top:1px solid #996;"|Total
 +
|style="border-top:1px solid #996;"|20 uL
 +
|style="border-top:1px solid #996;"|20 uL
 +
|style="border-top:1px solid #996;"|20 uL
 +
|style="border-top:1px solid #996; border-right:1px solid #996;"|20 uL
 +
|style="border-top:1px solid #996;"|20 uL
 +
|style="border-top:1px solid #996;"|20 uL
 +
|style="border-top:1px solid #996;"|20 uL
 +
|style="border-top:1px solid #996; border-right:1px solid #996;"|20 uL
 +
|style="border-top:1px solid #996;"|20 uL
 +
|style="border-top:1px solid #996;"|20 uL
 +
|style="border-top:1px solid #996;"|20 uL
 +
|style="border-top:1px solid #996;"|20 uL
 +
|}
 +
[[Image:HokkaidoU Japan 20100830a.jpg‎|200px|right|thumb|Electrophoresis of digestion products]]
 +
→Incubated at 37C for 60 min
 +
===Electrophoresis===
 +
* Markers used [https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''dIII,　EcoR I] and 50bp ladder
 +
* Was obvious that parts were cut as intended

Latest revision as of 07:59, 27 October 2010

Notebook

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

Electrophoresis

Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/August30"