Team:HokkaidoU Japan/Notebook/aggregation Week 11

From 2012.igem.org

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==September 10th==
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<div>
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==Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2==
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<p>
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To make a construct of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 by 3piece ligation, we digested pBAD-RBS-eCFP-RBS-pSB1A2 with EcoRI & SpeI, Ag43-dT-pSB1AK3 with XbaI & NotI and pSTV28 with EcoRI and NotI. Then we digested pT7-RBS-pSB1C3 with XbaI & SpeI as a control for confirmation of the ability of restriction enzyme.
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Insert1 (pBAD-RBS-eCFP-RBS-pSB1A2)
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{|class="hokkaidou-table-digestion"
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  |-
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  |DNA solution ( 30ng/ul)
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  |22 ul
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  |-
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  |XbaI
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  |1 ul
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  |-
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  |PstI
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  |1 ul
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  |-
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  |10xM buffer
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  |3 ul
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  |-
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  |DW
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  |3 ul
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  |-
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  |Total
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  |30 ul
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  |}
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Vector(pBAD-RBS-pSB1A2)
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{|class="hokkaidou-table-digestion"
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  |-
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  |DNA solution ( 50ng/ul)
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  |3 ul
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  |-
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  |SpeI
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  |1 ul
 +
  |-
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  |PstI
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  |1 ul
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  |-
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  |10xH buffer
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  |2 ul
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  |-
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  |DW
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  |13 ul
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  |-
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  |Total
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  |20 ul
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  |}
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control (eCFP-pSB1A2)
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{|class="hokkaidou-table-digestion"
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  |-
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  |DNA solution (30 ng/ul)
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  |5 ul
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  |-
 +
  |XbaI
 +
  |1 ul
 +
  |-
 +
  |SpeI
 +
  |1 ul
 +
  |-
 +
  |10xM buffer
 +
  |2 ul
 +
  |-
 +
  |DW
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  |11 ul
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  |-
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  |Total
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  |20 ul
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  |}
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{|class="hokkaidou-table-digestion"
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|-
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|Number
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|Degree
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|Minute
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|-
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|1
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|37
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|120
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|-
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|2
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|60
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|15
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|-
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|3
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|4
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|HOLD
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|}
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Revision as of 05:04, 10 September 2012

September 10th

Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2

To make a construct of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 by 3piece ligation, we digested pBAD-RBS-eCFP-RBS-pSB1A2 with EcoRI & SpeI, Ag43-dT-pSB1AK3 with XbaI & NotI and pSTV28 with EcoRI and NotI. Then we digested pT7-RBS-pSB1C3 with XbaI & SpeI as a control for confirmation of the ability of restriction enzyme. Insert1 (pBAD-RBS-eCFP-RBS-pSB1A2)

DNA solution ( 30ng/ul) 22 ul
XbaI 1 ul
PstI 1 ul
10xM buffer 3 ul
DW 3 ul
Total 30 ul



Vector(pBAD-RBS-pSB1A2)

DNA solution ( 50ng/ul) 3 ul
SpeI 1 ul
PstI 1 ul
10xH buffer 2 ul
DW 13 ul
Total 20 ul


control (eCFP-pSB1A2)

DNA solution (30 ng/ul) 5 ul
XbaI 1 ul
SpeI 1 ul
10xM buffer 2 ul
DW 11 ul
Total 20 ul


Number Degree Minute
1 37 120
2 60 15
3 4 HOLD

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