Team:HokkaidoU Japan/Notebook/September14

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{{Template:HokkaidoU_Japan}}<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September13|September 13]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September15|September 15]]</div></div>
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=Digestion and ligation of pSB1C3, araC Promoter and GFP=
 +
 
 +
==Digestion==
 +
 
 +
[[Image:HokkaidoU Japan 20100914a.jpg‎|200px|right|thumb|Electrophoresed digestion product]]
 +
 
 +
Used pSB1C3 which was PCRed september 6th. DNA concentration was 2256 ng/uL. So diluted 20 times.
 +
 
 +
{|style="text-align:center; float:left;" class="protocol"
 +
!Reagent
 +
!Amount
 +
|-
 +
|10x M buffer
 +
|5 uL
 +
|-
 +
|DW
 +
|34
 +
|-
 +
|pSB1C3
 +
|1
 +
|-
 +
|BSA
 +
|5
 +
|-
 +
|EcoR I
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|2
 +
|-
 +
|Pst I
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|3
 +
|-
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|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #996;"|'''50 uL'''
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|}
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{|style="text-align:center; float:left;" class="protocol"
 +
!Reagent
 +
!Amount
 +
|-
 +
|10x M buffer
 +
|5 uL
 +
|-
 +
|DW
 +
|27
 +
|-
 +
|Promoter
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|8
 +
|-
 +
|BSA
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|5
 +
|-
 +
|EcoR I
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|3
 +
|-
 +
|Spe I
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|2
 +
|-
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|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #996;"|'''50 uL'''
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|}
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{|style="text-align:center; float:left;" class="protocol"
 +
!Reagent
 +
!Amount
 +
|-
 +
|10x M buffer
 +
|5 uL
 +
|-
 +
|DW
 +
|32
 +
|-
 +
|GFP
 +
|3
 +
|-
 +
|BSA
 +
|5
 +
|-
 +
|Xba I
 +
|4
 +
|-
 +
|Pst I
 +
|1
 +
|-
 +
|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #996;"|'''50 uL'''
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|}
 +
 
 +
<div style="clear:both"></div>
 +
→Incubated at 37C for 120 min<br>
 +
→Added Sample Buffer 10 uL and electrophoresed
 +
* pSB1C3 band wasn't visible
 +
→ Other 2 were gel extracted
 +
* Diluted that final volume would be 40 uL
 +
 
 +
==[[Team:HokkaidoU_Japan/Protocols|Ethanol precipitation]]==
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[[Image:HokkaidoU Japan 20100914b.jpg‎|200px|right|thumb|Electrophoresis after ethanol precipitation]]
 +
 
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* Added ethanol acetate[3 M] 4 uL to 40 uL of previously purified DNA solution
 +
* Added EtOH[100%] 44 uL froze in dry ice
 +
* Melted and centrifuged at 15000 rpm, 4C 5 min
 +
* Discarded supernatant and added EtOH[70%] 100 uL rinsed
 +
* Centrifuged at 15,996rpm, 4C 5 min
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* Discarded supernatant and dried
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* Diluted in 2 uL of TE each
 +
 
 +
*Anticipated concentrations<br>
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promoter: 125 ng/2uL<br>
 +
GFP: 106 ng/2uL
 +
 
 +
* pSB1C3 was cut(EcoR1, Pst1) on September 6th
 +
* Did ethanol precipitation and electrophoresed to check concentration
 +
* It was about 20 ng/uL
 +
→Used 2 uL of it
 +
 
 +
==Ligation==
 +
 
 +
{|style="text-align:center;" class="protocol"
 +
!Reagent
 +
!Amount
 +
|-
 +
|pSB1C3
 +
|2 uL
 +
|-
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|promoter
 +
|2 uL
 +
|-
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|GFP
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|2 uL
 +
|-
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|Mighty Mix
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|6 uL
 +
|-
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|T4 ligase
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|0.4 uL
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|-
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|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #996;"|'''12.4 uL'''
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|}
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 +
*50 uL were transformed by electroporation and remaining 50 uL by heat shock (chemical)

Latest revision as of 08:20, 27 October 2010

Notebook

Digestion and ligation of pSB1C3, araC Promoter and GFP

Digestion

Electrophoresed digestion product

Used pSB1C3 which was PCRed september 6th. DNA concentration was 2256 ng/uL. So diluted 20 times.

Reagent Amount
10x M buffer 5 uL
DW 34
pSB1C3 1
BSA 5
EcoR I 2
Pst I 3
Total 50 uL
Reagent Amount
10x M buffer 5 uL
DW 27
Promoter 8
BSA 5
EcoR I 3
Spe I 2
Total 50 uL
Reagent Amount
10x M buffer 5 uL
DW 32
GFP 3
BSA 5
Xba I 4
Pst I 1
Total 50 uL

→Incubated at 37C for 120 min
→Added Sample Buffer 10 uL and electrophoresed

  • pSB1C3 band wasn't visible

→ Other 2 were gel extracted

  • Diluted that final volume would be 40 uL

Ethanol precipitation

Electrophoresis after ethanol precipitation
  • Added ethanol acetate[3 M] 4 uL to 40 uL of previously purified DNA solution
  • Added EtOH[100%] 44 uL froze in dry ice
  • Melted and centrifuged at 15000 rpm, 4C 5 min
  • Discarded supernatant and added EtOH[70%] 100 uL rinsed
  • Centrifuged at 15,996rpm, 4C 5 min
  • Discarded supernatant and dried
  • Diluted in 2 uL of TE each
  • Anticipated concentrations

promoter: 125 ng/2uL
GFP: 106 ng/2uL

  • pSB1C3 was cut(EcoR1, Pst1) on September 6th
  • Did ethanol precipitation and electrophoresed to check concentration
  • It was about 20 ng/uL

→Used 2 uL of it

Ligation

Reagent Amount
pSB1C3 2 uL
promoter 2 uL
GFP 2 uL
Mighty Mix 6 uL
T4 ligase 0.4 uL
Total 12.4 uL
  • 50 uL were transformed by electroporation and remaining 50 uL by heat shock (chemical)
Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September14"