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- | ==July 9th==
| |
- | <div class="hokkaidou-notebook-daily">
| |
- | 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.
| |
- | ;Colony PCR
| |
- | Colony PCR for assembly products.Each product reacted recipes written below.
| |
- | #picked up each 12(16 is best but there were only 12 colonies) colonies from LB plates by Autoclaved toothpicks.
| |
- | #Dipped into 10ul DW in 1.5ml eppendorf tubes.
| |
- | #from 10ul DW, 4ul was added in PCR reaction solution (below), 6ul was mixed with 200ul LB.
| |
- | #Ran PCR machine in recipe below.
| |
- | #Electrophoresis for confirmation of PCR results.
| |
| | | |
- |
| |
- | PCR reaction solution
| |
- | {|class="hokkaidou-table-pcr-reagent"
| |
- | |-
| |
- | |DNA solution
| |
- | |4ul
| |
- | |-
| |
- | |KapaTaq ready mix
| |
- | |5ul
| |
- | |-
| |
- | |BioBrick prefix forward primer
| |
- | |0.5ul
| |
- | |-
| |
- | |BioBrick suffix reverse primer
| |
- | |0.5ul
| |
- | |-
| |
- | |Total
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- | |10ul
| |
- | |}
| |
- |
| |
- |
| |
- | '''PCR recipe'''
| |
- |
| |
- | (pT7 + RBS)
| |
- | {|class="hokkaidou-table-pcr-time"
| |
- | |-
| |
- | |Number
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- | |Degree
| |
- | |Second
| |
- | |-
| |
- | |1
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- | |94
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- | |120
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- | |-
| |
- | |2
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- | |94
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- | |30
| |
- | |-
| |
- | |3
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- | |68
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- | |60
| |
- | |-
| |
- | |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.
| |
- | {|class="hokkaidou-table-pcr-time"
| |
- | |-
| |
- | |Number
| |
- | |Degree
| |
- | |Second
| |
- | |-
| |
- | |1
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- | |94
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- | |120
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- | |-
| |
- | |2
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- | |94
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- | |30
| |
- | |-
| |
- | |3
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- | |68
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- | |180
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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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- |
| |
- |
| |
- | ;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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- |
| |
- | [[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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- |
| |
- |
| |
- | ;Liquid culturing
| |
- | Liquid culture for mini-prep((pT7 + RBS) on pSB1K3 and (Ag43 + dT) on pSB1AK3).
| |
- | #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)).
| |
- | #Cultivated 15hrs30min.
| |
- | </div>
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- |
| |
- |
| |
- | ==July 10th==
| |
- | <div class="hokkaidou-notebook-daily">
| |
- |
| |
- | ;Mini-prep
| |
- | 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.
| |
- | #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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- |
| |
- | [[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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- |
| |
- | [[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.
| |
- |
| |
- | ;Digestion
| |
- | 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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- | |-
| |
- | |EcoRI
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- | |1ul
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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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- | |-
| |
- | |DW
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- | |14.5ul
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- | |-
| |
- | |Total
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- | |20ul
| |
- | |}
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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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- | |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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- | |-
| |
- | |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]
| |
- |
| |
- | ;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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- | |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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- | |-
| |
- | |10xH buffer
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- | |3ul
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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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- | Ag43-dT
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- | {|class="hokkaidou-table-digestion"
| |
- | |-
| |
- | |DNA
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- | |12.5ul
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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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- | |-
| |
- | |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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- | |DNA
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- | |20ul
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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.
| |
- | </div>
| |
- |
| |
- | ==July 11th==
| |
- | <div class="hokkaidou-notebook-daily">
| |
- |
| |
- | ;Ligation
| |
- | Ligation of pT7-RBS + Ag43-dT + pSB1C3(3A Assembly)
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- |
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- |
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- | Ligation recipe
| |
- | {|class="hokkaidou-table-ligation"
| |
- | |-
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- | |pT7-RBS
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- | |2ul
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- | |-
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- | |Ag43-dT
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- | |2ul
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- | |-
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- | |pSB1C3
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- | |3ul
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- | |-
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- | |Ligation Mighty Mix(TAKARA)
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- | |8ul
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- | |-
| |
- | |Total
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- | |16ul
| |
- | |}
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- |
| |
- |
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- | Ligation reaction time was written below.
| |
- |
| |
- | {|class="hokkaidou-table-ligation"
| |
- | |-
| |
- | |Degree
| |
- | |Minute
| |
- | |-
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- | |16
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- | |30
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- | |-
| |
- | |65
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- | |10
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- | |-
| |
- | |4
| |
- | |Hold
| |
- | |}
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- |
| |
- |
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- | Electrophoresis result
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- |
| |
- | [[image:HokkaidoU2012 120711 pt7-rbs-ag43-dtjpg.jpg]]
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- |
| |
- |
| |
- | ;Transformation
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- | Transformation for pT7-RBS + Ag43-dT + pSB1C3 into BL21(DE3)(E.coli which have T7 polymerase coadin site in their genomic DNA).
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- | #Added DNA soltions (Ligation products) 1ul to BL21(DE3) compitent cell.
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- | #Stood on ice in 30min.
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- | #Heatshock for 1min at 42c.
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- | #Added 200ul of LB to transformed BL21(DE3) solution.
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- | #Pre-cultivate in 2hrs
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- | #Splead 200ul of LB&BL21(DE3) solution supernant to LBC.
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- | #50ul of LB&BL21(DE3) solution were added to 200ul LB solution then spread 200ul to LBC plate.
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- | #Cultivated.
| |
- |
| |
- | </div>
| |
- |
| |
- | ==July 12th==
| |
- | <div class="hokkaidou-notebook-daily">
| |
- |
| |
- | ;Colony PCR
| |
- | Colony PCR for ligation product.Each products were reacted in recipes written below.
| |
- | #picked up each 16 colonies from LB plates by Autoclaved toothpicks.
| |
- | #Dipped into 10ul DW in 1.5ml eppendorf tubes.
| |
- | #from 10ul DW, 4ul was added in PCR reaction solution (below), 6ul was mixed with 200ul LB.
| |
- | #Ran PCR machine in recipe below.
| |
- | #Electrophoresis for confirmation of PCR results.
| |
- |
| |
- |
| |
- | PCR reaction solution
| |
- | {|class="hokkaidou-table-pcr-reagent"
| |
- | |-
| |
- | |DNA solution
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- | |4ul
| |
- | |-
| |
- | |KapaTaq ready mix
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- | |5ul
| |
- | |-
| |
- | |BioBrick prefix forward primer
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- | |0.5ul
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- | |-
| |
- | |BioBrick suffix reverse primer
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- | |0.5ul
| |
- | |-
| |
- | |Total
| |
- | |10ul
| |
- | |}
| |
- |
| |
- |
| |
- | '''PCR recipe'''
| |
- |
| |
- | (pT7 + RBS)
| |
- | {|class="hokkaidou-table-pcr-time"
| |
- | |-
| |
- | |Number
| |
- | |Degree
| |
- | |Second
| |
- | |-
| |
- | |1
| |
- | |94
| |
- | |120
| |
- | |-
| |
- | |2
| |
- | |94
| |
- | |30
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- | |-
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- | |3
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- | |68
| |
- | |90
| |
- | |-
| |
- | |4
| |
- | |4
| |
- | |HOLD
| |
- | |}
| |
- | Cycle:2~3 x 40
| |
- |
| |
- |
| |
- | ;Liquid culturing
| |
- | Liquid culture for some colonies used in colony PCR.
| |
- | #Prepared 200ul LB solutions.
| |
- | #To these LB solutions, added 6ul of LB solutions (colony PCR solutions were pre-cultivated in about 3hrs) and added 2ml LB and antibiotic(Cp).
| |
- | #Cultivated 18hrs30min.
| |
- |
| |
- |
| |
- | ==July 13th==
| |
- | <div class="hokkaidou-notebook-daily">
| |
- |
| |
- | ;Mini-prep
| |
- | Mini-prep for colony No.1~8
| |
- | We used mini-prep kit FastGene Plasmid Mini Kit (Nippon Genetics).
| |
- |
| |
- |
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- | Mini-prep result
| |
- |
| |
- | [[image:HokkaidoU2012 120713 pT7-RBS-Ag43-dT on pSB1C3 mini-prep.jpg]]
| |
- |
| |
- |
| |
- | ;Digestion
| |
- | Digestion to confirm how DNA fragments ligated.
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- |
| |
- |
| |
- | Digestion Recipe
| |
- | {|class="hokkaidou-table-digestion"
| |
- | |-
| |
- | |DNA
| |
- | |4ul
| |
- | |-
| |
- | |EcoRI
| |
- | |0.5ul
| |
- | |-
| |
- | |PstI
| |
- | |0.5ul
| |
- | |-
| |
- | |10xH buffer
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- | |2ul
| |
- | |-
| |
- | |DW
| |
- | |13ul
| |
- | |-
| |
- | |Total
| |
- | |20ul
| |
- | |}
| |
- |
| |
- |
| |
- | Digestion result
| |
- |
| |
- | [[image:HokkaidoU2012 120714 pT7-RBS-Ag43-dT on pSB1C3 digeation EP .jpg]]
| |
- |
| |
- |
| |
- | ==July 14th==
| |
- | <div class="hokkaidou-notebook-daily">
| |
- |
| |
- |
| |
- | Blue Screen!!!!
| |
- | Blue Screen!!!!
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- | Blue Screen!!!!
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- | Blue Screen!!!!
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- | Blue Screen!!!!
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- | Blue Screen!!!!
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- | Blue Screen!!!!
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- | Blue Screen!!!!
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- | Blue Screen!!!!
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- | Blue Screen!!!!
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- | Blue Screen!!!!
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- | Blue Screen!!!!
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- | Blue Screen!!!!
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- | Blue Screen!!!!
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- | Blue Screen!!!!
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- | Blue Screen!!!!
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- | Blue Screen!!!!
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- | Blue Screen!!!!
| |
- | Blue Screen!!!!
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- | Blue Screen!!!!
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- | Blue Screen!!!!
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- | Blue Screen!!!!
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- | Blue Screen!!!!
| |
- | Blue Screen!!!!
| |
- | Blue Screen!!!!
| |
- | Blue Screen!!!!
| |
- | Blue Screen!!!!
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- | Blue Screen!!!!
| |
- | Blue Screen!!!!
| |
- | Blue Screen!!!!
| |
- | Blue Screen!!!!
| |
- | Blue Screen!!!!
| |
- | Blue Screen!!!!
| |
- |
| |
- |
| |
- | ;Ligation
| |
- | Ligation for digestion fragments written above.
| |
- |
| |
- | Ligation recipe
| |
- | Each DNA fragments (pT7-RBS + pSB1C3 and Ag43-dT + pSB1T3) were reacted in recipe written below.
| |
- |
| |
- |
| |
- | {|class="hokkaidou-table-ligation"
| |
- | |-
| |
- | |Insert
| |
- | |5ul
| |
- | |-
| |
- | |Vector
| |
- | |1ul
| |
- | |-
| |
- | |Ligation Mighty Mix(TAKARA)
| |
- | |6ul
| |
- | |-
| |
- | |Total
| |
- | |12ul
| |
- | |}
| |
- |
| |
- |
| |
- | {|class="hokkaidou-table-ligation"
| |
- | |-
| |
- | |Degree
| |
- | |Minute
| |
- | |-
| |
- | |16
| |
- | |30
| |
- | |-
| |
- | |65
| |
- | |10
| |
- | |-
| |
- | |4
| |
- | |Hold
| |
- | |}
| |
- |
| |
- | Ligation result
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- | with colony no.1 (see colony pcr result in 9th)
| |
- |
| |
- | [[image:HokkaidoU2012 120714 pT7-RBS on 1K3 pSB1C3 Ag43-dT on 1AK3 pSB1T3 Ligation1,2 coloP-No.jpg]]
| |
- |
| |
- | ;Transformation
| |
- | Transformation for ligation products written above.
| |
- |
| |
- |
| |
- | #Added DNA soltions (Ligation products) 1ul to DH5α compitent cell.
| |
- | #Stood on ice in 30min.
| |
- | #Added 600ul of LB to transformed DH5α solution.
| |
- | #Pre-cultivate in 2hrs
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- | #Splead 300ul of LB&DH5α solution to LBC and LBT , 100ul added into 900ul of LB.
| |
- | #Splead 300ul of LB&DH5α solution from 1000ul LB (100ul added into 900ul) to LBC and LBT.
| |
- | #Cultivated.
| |
- |
| |
- |
| |
- |
| |
- | and to get more conformation about pT7-RBS-Ag43-dT on pSB1C3 was really ligated, we tried digest this DNA with EcoRI and PstI once more time.
| |
- |
| |
- | result
| |
- |
| |
- | [[image:HokkaidoU2012 120715 pt7-rbs-ag43-dt print.jpg]]
| |
- |
| |
- |
| |
- | ;Liquid culture
| |
- | Liquid culture for Ag43(BBa_K346007)
| |
- | #Picked up one colony from single colony isolated plate.
| |
- | #Dipped into LBC.
| |
- | #cultivated.
| |