Team:HokkaidoU Japan/Notebook/September6

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(Difference between revisions)
 
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=Ligation of pSB1C3 PCRed from [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-3A]] and [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|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)=
* Purified through gel extraction to remove primers
* Purified through gel extraction to remove primers
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!Amount
!Amount
|-
|-
-
|RFP ([[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-5A]])
+
|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
+
|10x '''M''' Buffer
|5 uL
|5 uL
|-
|-
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|5 uL
|5 uL
|-
|-
-
|10x M Buffer
+
|10x '''M''' Buffer
|5 uL
|5 uL
|-
|-
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!Amount
!Amount
|-
|-
-
|RFP ([[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-5A]])
+
|RFP ([[Team:HokkaidoU_Japan/Parts#BioBricks|1-5A]])
|0.75 uL
|0.75 uL
|-
|-

Latest revision as of 08:08, 27 October 2010

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