Team:HokkaidoU Japan/Notebook/September6

From 2010.igem.org

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=1-3A由来のpSB1C3と1-5A（RFP reporter）=
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=Ligation of pSB1C3 PCRed from [[Team:HokkaidoU_Japan/Parts#BioBricks|1-3A]] and [[Team:HokkaidoU_Japan/Parts#BioBricks|1-5A]]（RFP reporter）=
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* 制限酵素処理をする前にプライマーなどを確実に除くため，PCR productsの[[ゲル抽出]]を行った
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* 次の組成で制限酵素処理を行った
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* Purified through gel extraction to remove primers
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* Digested as listed in the table below
 +
 
{|style="text-align:center; float: left;" class="protocol"
{|style="text-align:center; float: left;" class="protocol"
!Reagent
!Reagent
!Amount
!Amount
|-
|-
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|RFP (1-5A)
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|RFP ([[Team:HokkaidoU_Japan/Parts#BioBricks|1-5A]])
|0.75 uL
|0.75 uL
|-
|-
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|5 uL
|5 uL
|-
|-
-
|10x M Buffer
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|10x '''M''' Buffer
|5 uL
|5 uL
|-
|-
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|5 uL
|5 uL
|-
|-
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|10x M Buffer
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|10x '''M''' Buffer
|5 uL
|5 uL
|-
|-
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!Amount
!Amount
|-
|-
-
|RFP (1-5A)
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|RFP ([[Team:HokkaidoU_Japan/Parts#BioBricks|1-5A]])
|0.75 uL
|0.75 uL
|-
|-
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|style="border-top:1px solid #996;"|'''50 uL'''
|style="border-top:1px solid #996;"|'''50 uL'''
|}
|}
 +
<div style="clear:both;"></div>
<div style="clear:both;"></div>
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* これらをゲル抽し，25 uLのDNase free water
+
 
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* この時点でそれぞれ22.56 ng/uL，45.12 ng/uLとなっていると見積もられる
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* Extracted from filter with  25 uL, 50 uL DNase free water
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* pSB1C3 2 uL, RFP 3 uLにMighty Mix 5 uLとT4 ligase 0.5 uLを加え，Ligationした
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* At this point solution volumes were 22.56 ng/uL，45.12 ng/uL acordingly
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* 50 uLのコンピテントセルをつかいTransformationした
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* Added 5 uL Mighty Mix and 0.5 uL T4 ligase to pSB1C3 2 uL, and RFP 3 uL
 +
* Ligated
 +
* [[Team:HokkaidoU_Japan/Protocols|Transformed]] to 50 uL of competent cells

Latest revision as of 08:08, 27 October 2010

Notebook

Ligation of pSB1C3 PCRed from 1-3A and 1-5A（RFP reporter）

  • Purified through gel extraction to remove primers
  • Digested as listed in the table below
Reagent Amount
RFP (1-5A) 0.75 uL
DW 34.3 uL
0.1% BSA 5 uL
10x M Buffer 5 uL
EcoR I 2.5 uL
Pst I 2.5 uL
Total 50 uL
Reagent Amount
pSB1C3 0.75 uL
DW 34.3 uL
0.1% BSA 5 uL
10x M Buffer 5 uL
EcoR I 2.5 uL
Pst I 2.5 uL
Total 50 uL
Reagent Amount
RFP (1-5A) 0.75 uL
DW 34.3 uL
0.1% BSA 5 uL
10x H Buffer 5 uL
EcoR I 2.5 uL
Pst I 2.5 uL
Total 50 uL
Reagent Amount
pSB1C3 0.75 uL
DW 34.3 uL
0.1% BSA 5 uL
10x H Buffer 5 uL
EcoR I 2.5 uL
Pst I 2.5 uL
Total 50 uL
  • Extracted from filter with 25 uL, 50 uL DNase free water
  • At this point solution volumes were 22.56 ng/uL，45.12 ng/uL acordingly
  • Added 5 uL Mighty Mix and 0.5 uL T4 ligase to pSB1C3 2 uL, and RFP 3 uL
  • Ligated
  • Transformed to 50 uL of competent cells
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