Team:HokkaidoU Japan/Notebook/August16

From 2010.igem.org

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{{Template:HokkaidoU_Japan}}
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{{Template:HokkaidoU_Japan}}<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/August13|August 13]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/August17|August 17]]</div></div>
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=制限酵素の活性チェック=
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==制限酵素処理==
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=Restriction Enzyme Activity Check=
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<div style="float:left; margin-left:20px;">
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==Restriction Enzyme Digestion==
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===EcoR I, Xba I, Pst I===
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<div style="float:left;">
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{|style="text-align: center;"
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 +
'''EcoR I, Xba I, Pst I'''
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 +
{|style="text-align: center;" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
|-
|-
|pUC119
|pUC119
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|1 uL
|1 uL
|-
|-
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|style="border-top:1px solid #000"|'''Total'''
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|style="border-top:1px solid #996"|'''Total'''
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|style="border-top:1px solid #000"|'''10 uL'''
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|style="border-top:1px solid #996"|'''10 uL'''
|}
|}
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* EcoR I, Xba I, Pst Iそれぞれ作る
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→37℃，60 min
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* Made solution for each EcoR I, Xba I, Pst I
 +
** incubated at 37C for 60 min
</div>
</div>
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<div style="float:left; margin-left:100px;">
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<div style="float:left;">
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===Spe I===
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'''Spe I'''
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{|style="text-align: center;"
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{|style="text-align: center;" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
|-
|-
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|1-1A
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|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-1A]]
|1 uL
|1 uL
|-
|-
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|1 uL
|1 uL
|-
|-
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|style="border-top:1px solid #000"|'''Total'''
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|style="border-top:1px solid #996"|'''Total'''
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|style="border-top:1px solid #000"|'''10 uL'''
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|style="border-top:1px solid #996"|'''10 uL'''
|}
|}
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* pUC119はSpe Iで切れないので，mini prepで得たプラスミドで確かめる
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* pUC119 doesn't have Spe I restriction sites, so we used mini preped plasmid with biobrick insert
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→37℃，60 min
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** incubated at 37C for 60 min
</div>
</div>
<div style="clear:both;"></div>
<div style="clear:both;"></div>
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===コントロール===
 
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* 1 uLのpUC119に? を加えた
 
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* 1 uLの1-1Aに？を加えた
 
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→37℃，60 min
 
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==電気泳動==
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===Control===
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[[Image:HokkaidoU Japan 20100816a.jpg‎|200px|right|thumb|]]
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* Added 1 uL of 6x Sample Buffer to 1 uL of pUC119
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制限酵素処理した溶液に2 uLの6x Sample Bufferを加え，電気泳動した<br>
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* Also added 1 uL of 6x Sample Buffer to 1 uL of [[Team:HokkaidoU_Japan/Parts#BioBricks|1-1A]]
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{|style="margin-left:20px;"
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** Incubated at 37C for 60 min
 +
 
 +
==Electrophoresis==
 +
 
 +
[[Image:HokkaidoU Japan 20100816a.jpg‎|200px|right|thumb|Electrophoresis of digested DNA]]
 +
 
 +
Added 2 uL of 6x Sample Buffer to digested solution and performed electrophoresis<br>
 +
 
 +
{|class="protocol"
|-
|-
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|レーン
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!Lane
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|
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!DNA
|-
|-
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|１
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|1
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|λ/''Hin''dIII
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|[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''dIII]
|-
|-
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|２
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|2
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|pUC119 コントロール
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|pUC119 control
|-
|-
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|３
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|3
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|1-1A コントロール
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|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-1A]] control
|-
|-
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|４
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|4
|pUC119 + EcoR I
|pUC119 + EcoR I
|-
|-
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|５
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|5
|pUC119 + Xba I
|pUC119 + Xba I
|-
|-
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|６
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|6
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|1-1A + Spe I
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|[Team:HokkaidoU_Japan/Parts#BioBricks|[1-1A]] + Spe I
|-
|-
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|７
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|7
|pUC119 + Pst I
|pUC119 + Pst I
|-
|-
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|８
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|8
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|空き
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|Empty
|}
|}
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=ベクターの制限酵素処理 (EcoR I, Pst I)=
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=Restriction Enzyme (EcoR I, Pst I) Digestion of the vector=
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{|style="text-align: center; margin-left:20px;"
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 +
{|style="text-align: center;" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
|-
|-
|pSB1C3
|pSB1C3
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|1 uL
|1 uL
|-
|-
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|style="border-top:1px solid #000"|'''Total'''
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|style="border-top:1px solid #996"|'''Total'''
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|style="border-top:1px solid #000"|'''20 uL'''
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|style="border-top:1px solid #996"|'''20 uL'''
|}
|}
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=パーツの制限酵素処理 (Xba I, Pst I)=
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=Restriction Enzyme (Xba I, Pst I) Digestion of the Parts=
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<div style="float:left; margin-left:20px;">
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<div style="float:left;">
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===RBS===
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'''RBS'''
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{|style="text-align: center;"
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{|style="text-align: center;" class="protocol"
|-
|-
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|1-2M
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!Reagent
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!Amount
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|-
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|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-2M]]
|2.5 uL
|2.5 uL
|-
|-
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|1 uL
|1 uL
|-
|-
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|style="border-top:1px solid #000"|'''Total'''
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|style="border-top:1px solid #996"|'''Total'''
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|style="border-top:1px solid #000"|'''20 uL'''
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|style="border-top:1px solid #996"|'''20 uL'''
|}
|}
</div>
</div>
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<div style="float:left; margin-left:100px;">
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<div style="float:left; margin-left:50px;">
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===Terminator===
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'''Terminator'''
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{|style="text-align: center;"
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{|style="text-align: center;" class="protocol"
 +
|-
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!Reagent
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!Amount
|-
|-
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|1-23L
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|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]]
|1.5 uL
|1.5 uL
|-
|-
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|1 uL
|1 uL
|-
|-
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|style="border-top:1px solid #000"|'''Total'''
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|style="border-top:1px solid #996"|'''Total'''
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|style="border-top:1px solid #000"|'''20 uL'''
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|style="border-top:1px solid #996"|'''20 uL'''
|}
|}
</div>
</div>
<div style="clear:both;"></div>
<div style="clear:both;"></div>
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=パーツの制限酵素処理 (EcoR I, Spe I)=
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=Restriction Enzyme (EcoR I, Spe I) Digestion of the Parts=
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<div style="float:left; margin-left:20px;">
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<div style="float:left;">
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===Heat shock promotor===
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'''Heat shock promotor'''
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{|style="text-align: center;"
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{|style="text-align: center;" class="protocol"
|-
|-
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|3-1E
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!Reagent
 +
!Amount
 +
|-
 +
|[[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]]
|5 uL
|5 uL
|-
|-
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|0.3 uL
|0.3 uL
|-
|-
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|style="border-top:1px solid #000"|'''Total'''
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|style="border-top:1px solid #996"|'''Total'''
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|style="border-top:1px solid #000"|'''20 uL'''
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|style="border-top:1px solid #996"|'''20 uL'''
|}
|}
</div>
</div>
<div style="float:left; margin-left:50px;">
<div style="float:left; margin-left:50px;">
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===RFP===
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'''RFP'''
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{|style="text-align: center;"
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{|style="text-align: center;" class="protocol"
 +
|-
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!Reagent
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!Amount
|-
|-
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|1-18F
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|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-18F]]
|5 uL
|5 uL
|-
|-
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|0.3 uL
|0.3 uL
|-
|-
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|style="border-top:1px solid #000"|'''Total'''
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|style="border-top:1px solid #996"|'''Total'''
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|style="border-top:1px solid #000"|'''20 uL'''
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|style="border-top:1px solid #996"|'''20 uL'''
|}
|}
</div>
</div>
<div style="clear:both;"></div>
<div style="clear:both;"></div>
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* 制限酵素の活性チェックでSpe Iの活性が低かったので，あとから0.7 uL加えた
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* It was obvious after restriction enzyme activity check that Spe I was week, so additional 0.7 uL was added

Latest revision as of 07:31, 27 October 2010

Notebook

Restriction Enzyme Activity Check

Restriction Enzyme Digestion

EcoR I, Xba I, Pst I

Reagent Amount
pUC119 1 uL
DW 7 uL
10x M Buffer 1 uL
Restriction Enzyme 1 uL
Total 10 uL
  • Made solution for each EcoR I, Xba I, Pst I
    • incubated at 37C for 60 min

Spe I

Reagent Amount
1-1A 1 uL
DW 7 uL
10x M Buffer 1 uL
Restriction Enzyme 1 uL
Total 10 uL
  • pUC119 doesn't have Spe I restriction sites, so we used mini preped plasmid with biobrick insert
    • incubated at 37C for 60 min

Control

  • Added 1 uL of 6x Sample Buffer to 1 uL of pUC119
  • Also added 1 uL of 6x Sample Buffer to 1 uL of 1-1A
    • Incubated at 37C for 60 min

Electrophoresis

Electrophoresis of digested DNA

Added 2 uL of 6x Sample Buffer to digested solution and performed electrophoresis

Lane DNA
1 λ/HindIII
2 pUC119 control
3 1-1A control
4 pUC119 + EcoR I
5 pUC119 + Xba I
6 [1-1A]] + Spe I
7 pUC119 + Pst I
8 Empty

Restriction Enzyme (EcoR I, Pst I) Digestion of the vector

Reagent Amount
pSB1C3 4 uL
DW 10 uL
10x M Buffer 2 uL
BSA 2 uL
EcoR I 1 uL
Pst I 1 uL
Total 20 uL

Restriction Enzyme (Xba I, Pst I) Digestion of the Parts

RBS

Reagent Amount
1-2M 2.5 uL
DW 11.5 uL
10x M Buffer 2 uL
BSA 2 uL
Xba I 1 uL
Pst I 1 uL
Total 20 uL

Terminator

Reagent Amount
1-23L 1.5 uL
DW 12.5 uL
10x M Buffer 2 uL
BSA 2 uL
Xba I 1 uL
Pst I 1 uL
Total 20 uL

Restriction Enzyme (EcoR I, Spe I) Digestion of the Parts

Heat shock promotor

Reagent Amount
3-1E 5 uL
DW 9.7 uL
10x M Buffer 2 uL
BSA 2 uL
EcoR I 1 uL
Spe I 0.3 uL
Total 20 uL

RFP

Reagent Amount
1-18F 5 uL
DW 9.7 uL
10x M Buffer 2 uL
BSA 2 uL
EcoR I 1 uL
Spe I 0.3 uL
Total 20 uL
  • It was obvious after restriction enzyme activity check that Spe I was week, so additional 0.7 uL was added
Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/August16"