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| - | {{Team:HokkaidoU_Japan/header}}
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| - | {{Team:HokkaidoU_Japan/nav.notebook}}
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| - | <div id="hokkaidou-column-main">
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| - | <!-- DO NOT EDIT OVER THIS LINE @iTakeshi -->
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| - | ==July 16th==
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| - | <div class="hokkaidou-notebook-daily">
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| - | Ag43, dT
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| - | ===Digestion===
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| - | Results of digestion in 15th.
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| - | [[image:HokkaidoU2012 120716 Ag43 on pSB1C3 digestion with S&P-1.jpg|thumb|Digestion result image]]
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| - | Product:Ag43(K346007)=3120bp and 2070bp, pT7-RBS on pSB1K3=2247bp
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| - | We confirmed there are some kind of restriction enzyme site in K346007 (digested with SpeI, PstI) and pT7-RBS on pSB1K3 was successfully digested with EcoRI and PstI. Balance between d+(EcoRI) and d+(E&P:cut with EcoRI and PstI) is about 80bp.
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| - |
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| - |
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| - | ===Gel Extraction===
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| - | Gel Extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics).
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| - | Got 50ul of DNA solution.
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| - |
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| - |
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| - | ===Ethanol Precipitation===
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| - | Ethanol Precipitation for digestion and gel extraction products.
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| - | #Added 5ul of NaoAc, 1.5ul of glycogen and 125ul of 100% ethanol.
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| - | #Centrifuged in 15000rpm, 10min at 4C.
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| - | #Remove supernatant and added 220ul of 70% ethanol.
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| - | #Centrifuged in 15000rpm, 10min at 4C.
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| - | #Remove supernatant and air drying in room temperature then added 5ul of DW.
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| - |
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| - | ----
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| - | dT(B0015) would be amplified incorrectly. So we tried another DNA amplification method: PCR.
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| - |
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| - | ===PCR===
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| - | PCR for dT(B0015)
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| - |
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| - | {|class="hokkaidou-table-pcr-reagent"
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| - | |-
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| - | |DNA solution
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| - | |1ul
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| - | |-
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| - | |KOD-Plus-NEO(Taq polymerase)
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| - | |1ul
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| - | |-
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| - | |dNTP
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| - | |5ul
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| - | |-
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| - | |MgSO4
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| - | |3ul
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| - | |-
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| - | |KOD-Plus-NEO Buffer
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| - | |5ul
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| - | |-
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| - | |Forward Primer(100bp_up forward primer)
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| - | |1ul
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| - | |-
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| - | |Reverse Primer(200bp_down Reverse primer)
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| - | |1ul
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| - | |-
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| - | |DW
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| - | |33ul
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| - | |-
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| - | |Total
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| - | |50ul
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| - | |}
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| - |
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| - |
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| - | {|class="hokkaidou-table-pcr-time"
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| - | |-
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| - | |Number
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| - | |Degree
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| - | |Second
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| - | |-
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| - | |1
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| - | |94
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| - | |120
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| - | |-
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| - | |2
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| - | |98
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| - | |10
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| - | |-
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| - | |3
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| - | |58
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| - | |30
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| - | |-
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| - | |4
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| - | |68
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| - | |30
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| - | |-
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| - | |5
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| - | |4
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| - | |HOLD
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| - | |}
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| - | Cycle:2~4 x 45
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| - |
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| - |
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| - |
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| - | [[image:HokkaidoU2012 120716-dt-ethandpcr.jpg|thumb|PCR result image]]
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| - |
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| - | We migrated B0015 mini-prep psoduct, digestion product, and PCR product.
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| - | PCRed dT would have 429bp(100bp(added by forward primer) + 129bp(dT) + 200bp(added by reverse primer)). Most bright and thick band in this image has about 300~400 bp. We thought dT was amplified successfully.
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| - |
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| - |
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| - | ----
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| - |
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| - | Ag43
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| - |
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| - | Digestion result of Ag43 was incorrect. We digested Ag43 once more time.
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| - |
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| - | ===Digestion===
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| - | Digestion for Ag43 with SpeI and PstI.
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| - |
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| - | {|class="hokkaidou-table-digestion"
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| - | |-
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| - | |Ag43 DNA solution
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| - | |9ul
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| - | |-
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| - | |SpeI
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| - | |1ul
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| - | |-
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| - | |PstI
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| - | |1ul
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| - | |-
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| - | |10xH buffer
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| - | |2ul
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| - | |-
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| - | |DW
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| - | |7ul
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| - | |-
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| - | |Total
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| - | |20ul
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| - | |}
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| - |
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| - |
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| - | [[image:|thumb|Digestion result image]]
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| - |
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| - |
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| - | ===Liquid Culture===
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| - | Liquid culture for single colony isolated pT7-RBS on pSB1C3 and Ag43-dT on pSB1T3.
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| - | #Picked up one colony from single colony isolated plates by platinum loop.
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| - | #Dipped into 2ml of LBC and LBT.
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| - | #Cultivated.
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| - | <!-- DO NOT EDIT UNDER THIS LINE @iTakeshi -->
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| - | </div>
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| - | <br style="line-height: 0; clear: both;" />
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| - | {{Team:HokkaidoU_Japan/footer}}
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