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| - | ==9th==
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| - | <div class="hokkaidou-notebook-daily">
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| - | pT7 + RBS (3A Assembly) and Ag43 + dT (standard assembly) ligation products were transformed and cultivated then some colonies were formed (12~16 colonies) so we confirmed really insert DNA (3A:pT7 and RBS, standard:Ag43 and dT) were inserted to vector or not by colony PCR.
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| - | ;Colony PCR
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| - | Colony PCR for assembly products.Each product reacted recipes written below.
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| - | #picked up each 12(16 is best but there were only 12 colonies) colonies from LB plates by Autoclaved toothpicks.
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| - | #Dipped into 10ul DW in 1.5ml eppendorf tubes.
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| - | #from 10ul DW, 4ul was added in PCR reaction solution (below), 6ul was mixed with 200ul LB.
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| - | #Ran PCR machine in recipe below.
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| - | #Electrophoresis for confirmation of PCR results.
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| | | | |
| - |
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| - | PCR reaction solution
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| - | {|class="hokkaidou-table-pcr-reagent"
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| - | |-
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| - | |DNA solution
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| - | |4ul
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| - | |-
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| - | |KapaTaq ready mix
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| - | |5ul
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| - | |-
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| - | |BioBrick prefix forward primer
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| - | |0.5ul
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| - | |-
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| - | |BioBrick suffix reverse primer
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| - | |0.5ul
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| - | |-
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| - | |Total
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| - | |10ul
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| - | |}
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| - |
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| - |
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| - | '''PCR recipe'''
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| - |
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| - | (pT7 + RBS)
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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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| - | |94
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| - | |30
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| - | |-
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| - | |3
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| - | |68
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| - | |60
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| - | |-
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| - | |4
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| - | |4
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| - | |HOLD
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| - | |}
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| - | Cycle:2~3 x 40
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| - |
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| - |
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| - | (Ag43 + dT)
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| - |
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| - | Ag43 + dT (+ Biobrick prefis & suffix) is about 3290bp. Extension step needed >180seconds.
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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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| - | |94
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| - | |30
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| - | |-
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| - | |3
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| - | |68
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| - | |180
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| - | |-
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| - | |4
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| - | |4
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| - | |HOLD
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| - | |}
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| - | Cycle:2~3 x 35
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| - |
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| - |
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| - | ;Electrophoresis results
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| - | Electrophoresis for (pT7 + RBS) in 2% agarose gel and (Ag43 + dT) in 1% agarose gel.
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| - |
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| - |
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| - | pT7 + RBS on pSB1K3
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| - | bbp-Insert-bbs:86bp
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| - |
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| - | [[image:HokkaidoU2012 120709 pt7-rbs 75% scale.jpg]]
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| - |
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| - |
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| - | Ag43 + dT on pSB1AK3
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| - | bbp-Insert-bbs:3290bp
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| - |
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| - | [[image:HokkaidoU2012 120709 ag43-dt-1 75% scale.jpg]]
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| - |
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| - |
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| - | We couldn't confirm insert DNA were really ligated with Vector or not.
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| - | Next we tried confirmation of insert DNA by Electrophoresis of mini-prep products.
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| - | For mini-prep, we needed do liquid culture.
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| - |
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| - |
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| - | ;Liquid culturing
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| - | Liquid culture for mini-prep((pT7 + RBS) on pSB1K3 and (Ag43 + dT) on pSB1AK3).
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| - | #Prepared 1800ul LB solutions.
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| - | #To these LB solutions, added 200ul of LB solutions (colony PCR solutions were pre-cultivated in about 2hrs) and each antibiotics(Km (for (pT7 + RBS) on pSB1K3) and Amp(for (Ag43 + dT) on pSB1AK3)).
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| - | #Cultivated 15hrs30min.
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| - | </div>
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| - |
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| - |
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| - | ==10th==
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| - | <div class="hokkaidou-notebook-daily">
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| - |
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| - | ;Mini-prep
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| - | Mini-prep for some colonies (we selected colonies which showed more correct bands than other colonies, pT7+RBS were No.4, 5, 9, 10 colonies and Ag43 + dT were No.1, 2, 3, 4 colonies)cultivated in LBA(Ag43 + dT) and LBK(pT7 + RBS) in 15hrs 30min.
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| - | #Mini-prep for LB culturing products along the protocol of FastGene Plasmid Mini kit(Nippon Genetics).
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| - | #Electrophoresis (Used pre-migrated 1% agarose gels with 5ul EtBr)in 30min.
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| - |
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| - | Electrophoresis resulsts
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| - |
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| - | pT7 + RBS on pSB1K3(Total 2247bp)
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| - |
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| - | [[image:HokkaidoU2012 120710 miniprepproduct T7+RBS.jpg]]
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| - |
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| - |
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| - | Ag43 + dT on pSB1AK3(Total 6444bp)
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| - |
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| - | [[image:HokkaidoU2012 120710 miniprepproduct Ag43+dT.jpg]]
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| - |
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| - | To confirm more about insert DNA, we tried to digest these DNA with EcoRI and PstI.
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| - |
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| - | ;Digestion
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| - | Digestion for confirmation of insert DNA. Each DNA mini-prep products were digested with E & P.
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| - |
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| - |
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| - | Digestion recipe
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| - | pT7-RBS
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| - | {|class="hokkaidou-table-digestion"
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| - | |-
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| - | |pT7-RBS
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| - | |1,5ul
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| - | |-
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| - | |EcoRI
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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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| - | |14.5ul
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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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| - | Digestion recipe
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| - | Ag43-dT
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| - | {|class="hokkaidou-table-digestion"
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| - | |-
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| - | |Ag43-dT
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| - | |4ul
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| - | |-
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| - | |EcoRI
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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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| - | |12ul
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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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| - | Digestioned at 37c in 2hrs.
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| - |
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| - |
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| - | Digestion results
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| - |
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| - | pT7+RBS
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| - |
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| - | [[image:HokkaidoU2012 120710 pt7-rbs,digestion.jpg]]
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| - |
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| - |
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| - | Ag43+dT
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| - |
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| - | [[image:HokkaidoU2012 120710 ag43-dt,digestion.jpg]]
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| - |
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| - |
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| - | Insert DNA were correct we thought. We tried digestion for 3A Assembly[pT7-RBS + Ag43-dT + pSB1C3]
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| - |
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| - | ;Digestion
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| - | Digestion for 3A Assembly.
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| - |
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| - |
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| - | pT7-RBS
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| - | {|class="hokkaidou-table-digestion"
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| - | |-
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| - | |DNA
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| - | |17ul
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| - | |-
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| - | |EcoRI
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| - | |1ul
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| - | |-
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| - | |SpeI
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| - | |1ul
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| - | |-
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| - | |10xH buffer
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| - | |3ul
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| - | |-
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| - | |DW
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| - | |8ul
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| - | |-
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| - | |Total
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| - | |30ul
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| - | |}
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| - |
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| - |
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| - | Ag43-dT
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| - | {|class="hokkaidou-table-digestion"
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| - | |-
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| - | |DNA
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| - | |12.5ul
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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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| - | |10xH buffer
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| - | |2ul
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| - | |-
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| - | |DW
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| - | |3.5ul
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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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| - | pSB1C3
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| - | {|class="hokkaidou-table-digestion"
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| - | |-
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| - | |DNA
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| - | |20ul
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| - | |-
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| - | |EcoRI
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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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| - | |3ul
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| - | |-
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| - | |DW
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| - | |5ul
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| - | |-
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| - | |Total
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| - | |30ul
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| - | |}
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| - |
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| - | Reacted in 2hrs at 37c.
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| - | </div>
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