Team:HokkaidoU Japan/Notebook/August16

From 2010.igem.org

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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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=Restriction Enzyme Activity Check=
 +
==Restriction Enzyme Digestion==
 +
<div style="float:left;">
 +
 
 +
'''EcoR I, Xba I, Pst I'''
 +
 
 +
{|style="text-align: center;" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
 +
|-
 +
|pUC119
 +
|1 uL
 +
|-
 +
|DW
 +
|7 uL
 +
|-
 +
|10x M Buffer
 +
|1 uL
 +
|-
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|Restriction Enzyme
 +
|1 uL
 +
|-
 +
|style="border-top:1px solid #996"|'''Total'''
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|style="border-top:1px solid #996"|'''10 uL'''
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|}
 +
 
 +
* Made solution for each EcoR I, Xba I, Pst I
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** incubated at 37C for 60 min
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</div>
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<div style="float:left;">
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'''Spe I'''
 +
{|style="text-align: center;" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
 +
|-
 +
|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-1A]]
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|1 uL
 +
|-
 +
|DW
 +
|7 uL
 +
|-
 +
|10x M Buffer
 +
|1 uL
 +
|-
 +
|Restriction Enzyme
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|1 uL
 +
|-
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|style="border-top:1px solid #996"|'''Total'''
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|style="border-top:1px solid #996"|'''10 uL'''
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|}
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* pUC119 doesn't have Spe I restriction sites, so we used mini preped plasmid with biobrick insert
 +
** incubated at 37C for 60 min
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</div>
 +
<div style="clear:both;"></div>
 +
 
 +
===Control===
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* Added 1 uL of 6x Sample Buffer to 1 uL of pUC119
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* 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"
 +
|-
 +
!Lane
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!DNA
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|-
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|1
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|[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''dIII]
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|-
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|2
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|pUC119 control
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|-
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|3
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|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-1A]] control
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|-
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|4
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|pUC119 + EcoR I
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|-
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|5
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|pUC119 + Xba I
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|-
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|6
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|[Team:HokkaidoU_Japan/Parts#BioBricks|[1-1A]] + Spe I
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|-
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|7
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|pUC119 + Pst I
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|-
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|8
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|Empty
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|}
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=Restriction Enzyme (EcoR I, Pst I) Digestion of the vector=
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 +
{|style="text-align: center;" class="protocol"
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|-
 +
!Reagent
 +
!Amount
 +
|-
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|pSB1C3
 +
|4 uL
 +
|-
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|DW
 +
|10 uL
 +
|-
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|10x M Buffer
 +
|2 uL
 +
|-
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|BSA
 +
|2 uL
 +
|-
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|EcoR I
 +
|1 uL
 +
|-
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|Pst I
 +
|1 uL
 +
|-
 +
|style="border-top:1px solid #996"|'''Total'''
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|style="border-top:1px solid #996"|'''20 uL'''
 +
|}
 +
 
 +
=Restriction Enzyme (Xba I, Pst I) Digestion of the Parts=
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<div style="float:left;">
 +
'''RBS'''
 +
{|style="text-align: center;" class="protocol"
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|-
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!Reagent
 +
!Amount
 +
|-
 +
|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-2M]]
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|2.5 uL
 +
|-
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|DW
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|11.5 uL
 +
|-
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|10x M Buffer
 +
|2 uL
 +
|-
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|BSA
 +
|2 uL
 +
|-
 +
|Xba I
 +
|1 uL
 +
|-
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|Pst I
 +
|1 uL
 +
|-
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|style="border-top:1px solid #996"|'''Total'''
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|style="border-top:1px solid #996"|'''20 uL'''
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|}
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</div>
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<div style="float:left; margin-left:50px;">
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'''Terminator'''
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{|style="text-align: center;" class="protocol"
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|-
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!Reagent
 +
!Amount
 +
|-
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|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]]
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|1.5 uL
 +
|-
 +
|DW
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|12.5 uL
 +
|-
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|10x M Buffer
 +
|2 uL
 +
|-
 +
|BSA
 +
|2 uL
 +
|-
 +
|Xba I
 +
|1 uL
 +
|-
 +
|Pst I
 +
|1 uL
 +
|-
 +
|style="border-top:1px solid #996"|'''Total'''
 +
|style="border-top:1px solid #996"|'''20 uL'''
 +
|}
 +
</div>
 +
<div style="clear:both;"></div>
 +
 
 +
=Restriction Enzyme (EcoR I, Spe I) Digestion of the Parts=
 +
<div style="float:left;">
 +
'''Heat shock promotor'''
 +
{|style="text-align: center;" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
 +
|-
 +
|[[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]]
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|5 uL
 +
|-
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|DW
 +
|9.7 uL
 +
|-
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|10x M Buffer
 +
|2 uL
 +
|-
 +
|BSA
 +
|2 uL
 +
|-
 +
|EcoR I
 +
|1 uL
 +
|-
 +
|Spe I
 +
|0.3 uL
 +
|-
 +
|style="border-top:1px solid #996"|'''Total'''
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|style="border-top:1px solid #996"|'''20 uL'''
 +
|}
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</div>
 +
<div style="float:left; margin-left:50px;">
 +
'''RFP'''
 +
{|style="text-align: center;" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
 +
|-
 +
|[[Team:HokkaidoU_Japan/Parts#BioBricks|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
 +
|-
 +
|style="border-top:1px solid #996"|'''Total'''
 +
|style="border-top:1px solid #996"|'''20 uL'''
 +
|}
 +
</div>
 +
<div style="clear:both;"></div>
 +
* 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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