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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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| - | <div class="hokkaidou-notebook-daily">
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| - | ==July 16th==
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| - | <div>
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| - | Ag43, dT
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| - | ===Digestion===
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| - | <p>
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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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| - |
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| - | dT(B0015) would be amplified incorrectly and couldn't get enough digestion product. So we tried another DNA amplification method: PCR then digested.
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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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| - | ===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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| - | ===Digestion===
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| - | '''Digestion for dT which amplified with PCR.
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| - | Digested with XbaI and PstI'''.
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| - |
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| - | dT
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| - | {|class="hokkaidou-table-digestion"
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| - | |-
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| - | |DNA solution
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| - | |5ul
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| - | |-
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| - | |XbaI
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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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| - | |10xM buffer
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| - | |2ul
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| - | |-
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| - | |DW
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| - | |11ul
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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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| - | ----
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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:HokkaidoU2012 120716 Ag43d- Ag43d+.jpg|thumb|Digestion result image]]
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| - |
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| - | There are same results with digestion result of recent. '''We thought PstI would cut different site'''.
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| - | What is this 500bp fragment????
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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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| - |
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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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| - | </div>
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| - |
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| - |
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| - | <div class="hokkaidou-notebook-daily">
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| - | ==July 17th==
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| - | <div>
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| - | digestioned->Ethanol precipitation->Gel Extraction->Ethanol Precipitation
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| - |
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| - | ----
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| - | Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3
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| - | ===Mini-prep===
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| - | Mini-prep for liquid culture products cultivated from yesterday(16th).
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| - | We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50ul of DNA solutions.
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| - |
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| - | ===Electrophoresis===
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| - | Electrophoresis for digestion product of K346007(Ag43) with EcoRI and SpeI as a reference to confirm SpeI cuts correct site or not and PstI didn't work correctly(in past experiments). And mini-prep products of Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 also migrated.
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| - |
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| - |
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| - | If digestion were succeeded, there are two bands would be seen: about 3120bp and 2070bp.
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| - | And if mini-prep were succeeded, there would exist two bands in each lanes: about 5000bp and 2000bp.
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| - |
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| - | [[image:HokkaidoU2012 120717 Ag43-Ag43d+(E&S)-(Ag43-dT on pSB1T3)-(pT7-RBS on pSB1C3).jpg|thumb|Digestion and mini-prep result image]]
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| - |
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| - | In this result we confirmed Ag43 was successfully digested with EcoRI and SpeI, and there were no other extra bands which had been existed double digested with EcoRI & PstI and SpeI & PstI. '''would PstI not work correctly?'''
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| - |
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| - |
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| - | About mini-prep products,
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| - | </div>
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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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