Team:HokkaidoU Japan/Notebook/August16

From 2010.igem.org

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-
=制限酵素の活性チェック=
+
=Restriction Enzyme Activity Check=
-
==制限酵素処理==
+
==Restriction Enzyme Digestion==
<div style="float:left;">
<div style="float:left;">
 +
'''EcoR I, Xba I, Pst I'''
'''EcoR I, Xba I, Pst I'''
 +
{|style="text-align: center;" class="protocol"
{|style="text-align: center;" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
|-
|-
|pUC119
|pUC119
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|1 uL
|1 uL
|-
|-
-
|style="border-top:1px solid #000"|'''Total'''
+
|style="border-top:1px solid #996"|'''Total'''
-
|style="border-top:1px solid #000"|'''10 uL'''
+
|style="border-top:1px solid #996"|'''10 uL'''
|}
|}
-
* EcoR I, Xba I, Pst Iそれぞれ作る
+
 
-
→37℃，60 min
+
* Made solution for each EcoR I, Xba I, Pst I
 +
** incubated at 37C for 60 min
</div>
</div>
<div style="float:left;">
<div style="float:left;">
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{|style="text-align: center;" class="protocol"
{|style="text-align: center;" class="protocol"
|-
|-
-
|1-1A
+
!Reagent
 +
!Amount
 +
|-
 +
|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-1A]]
|1 uL
|1 uL
|-
|-
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|1 uL
|1 uL
|-
|-
-
|style="border-top:1px solid #000"|'''Total'''
+
|style="border-top:1px solid #996"|'''Total'''
-
|style="border-top:1px solid #000"|'''10 uL'''
+
|style="border-top:1px solid #996"|'''10 uL'''
|}
|}
-
* 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
+
* pUC119 doesn't have Spe I restriction sites, so we used mini preped plasmid with biobrick insert
-
→37℃，60 min
+
** incubated at 37C for 60 min
</div>
</div>
<div style="clear:both;"></div>
<div style="clear:both;"></div>
-
===コントロール===
 
-
* 1 uLのpUC119に1 uLの6x Sample Bufferを加えた
 
-
* 1 uLの1-1Aに1 uLの6x Sample Bufferを加えた
 
-
→37℃，60 min
 
-
==電気泳動==
+
===Control===
-
[[Image:HokkaidoU Japan 20100816a.jpg‎|200px|right|thumb|]]
+
* Added 1 uL of 6x Sample Buffer to 1 uL of pUC119
-
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>
+
* Also added 1 uL of 6x Sample Buffer to 1 uL of [[Team:HokkaidoU_Japan/Parts#BioBricks|1-1A]]
 +
** 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"
{|class="protocol"
|-
|-
-
|レーン
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!Lane
-
|
+
!DNA
|-
|-
-
|１
+
|1
-
|λ/''Hin''dIII
+
|[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''dIII]
|-
|-
-
|２
+
|2
-
|pUC119 コントロール
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|pUC119 control
|-
|-
-
|３
+
|3
-
|1-1A コントロール
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|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-1A]] control
|-
|-
-
|４
+
|4
|pUC119 + EcoR I
|pUC119 + EcoR I
|-
|-
-
|５
+
|5
|pUC119 + Xba I
|pUC119 + Xba I
|-
|-
-
|６
+
|6
-
|1-1A + Spe I
+
|[Team:HokkaidoU_Japan/Parts#BioBricks|[1-1A]] + Spe I
|-
|-
-
|７
+
|7
|pUC119 + Pst I
|pUC119 + Pst I
|-
|-
-
|８
+
|8
-
|空き
+
|Empty
|}
|}
-
=ベクターの制限酵素処理 (EcoR I, Pst I)=
+
=Restriction Enzyme (EcoR I, Pst I) Digestion of the vector=
 +
 
{|style="text-align: center;" class="protocol"
{|style="text-align: center;" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
|-
|-
|pSB1C3
|pSB1C3
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|1 uL
|1 uL
|-
|-
-
|style="border-top:1px solid #000"|'''Total'''
+
|style="border-top:1px solid #996"|'''Total'''
-
|style="border-top:1px solid #000"|'''20 uL'''
+
|style="border-top:1px solid #996"|'''20 uL'''
|}
|}
-
=パーツの制限酵素処理 (Xba I, Pst I)=
+
=Restriction Enzyme (Xba I, Pst I) Digestion of the Parts=
-
<div style="float:left; margin-left:20px;">
+
<div style="float:left;">
'''RBS'''
'''RBS'''
{|style="text-align: center;" class="protocol"
{|style="text-align: center;" class="protocol"
|-
|-
-
|1-2M
+
!Reagent
 +
!Amount
 +
|-
 +
|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-2M]]
|2.5 uL
|2.5 uL
|-
|-
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|1 uL
|1 uL
|-
|-
-
|style="border-top:1px solid #000"|'''Total'''
+
|style="border-top:1px solid #996"|'''Total'''
-
|style="border-top:1px solid #000"|'''20 uL'''
+
|style="border-top:1px solid #996"|'''20 uL'''
|}
|}
</div>
</div>
-
<div style="float:left; margin-left:100px;">
+
<div style="float:left; margin-left:50px;">
'''Terminator'''
'''Terminator'''
{|style="text-align: center;" class="protocol"
{|style="text-align: center;" class="protocol"
|-
|-
-
|1-23L
+
!Reagent
 +
!Amount
 +
|-
 +
|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]]
|1.5 uL
|1.5 uL
|-
|-
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|1 uL
|1 uL
|-
|-
-
|style="border-top:1px solid #000"|'''Total'''
+
|style="border-top:1px solid #996"|'''Total'''
-
|style="border-top:1px solid #000"|'''20 uL'''
+
|style="border-top:1px solid #996"|'''20 uL'''
|}
|}
</div>
</div>
<div style="clear:both;"></div>
<div style="clear:both;"></div>
-
=パーツの制限酵素処理 (EcoR I, Spe I)=
+
=Restriction Enzyme (EcoR I, Spe I) Digestion of the Parts=
-
<div style="float:left; margin-left:20px;">
+
<div style="float:left;">
'''Heat shock promotor'''
'''Heat shock promotor'''
{|style="text-align: center;" class="protocol"
{|style="text-align: center;" class="protocol"
|-
|-
-
|3-1E
+
!Reagent
 +
!Amount
 +
|-
 +
|[[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]]
|5 uL
|5 uL
|-
|-
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|0.3 uL
|0.3 uL
|-
|-
-
|style="border-top:1px solid #000"|'''Total'''
+
|style="border-top:1px solid #996"|'''Total'''
-
|style="border-top:1px solid #000"|'''20 uL'''
+
|style="border-top:1px solid #996"|'''20 uL'''
|}
|}
</div>
</div>
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{|style="text-align: center;" class="protocol"
{|style="text-align: center;" class="protocol"
|-
|-
-
|1-18F
+
!Reagent
 +
!Amount
 +
|-
 +
|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-18F]]
|5 uL
|5 uL
|-
|-
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|0.3 uL
|0.3 uL
|-
|-
-
|style="border-top:1px solid #000"|'''Total'''
+
|style="border-top:1px solid #996"|'''Total'''
-
|style="border-top:1px solid #000"|'''20 uL'''
+
|style="border-top:1px solid #996"|'''20 uL'''
|}
|}
</div>
</div>
<div style="clear:both;"></div>
<div style="clear:both;"></div>
-
* 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
+
* 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
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