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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">
| |
| - | ==July 9th==
| |
| - | <div>
| |
| - | 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===
| |
| - | <p>
| |
| - | 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
| |
| - | |10ul
| |
| - | |}
| |
| | | | |
| - |
| |
| - | PCR recipe
| |
| - | (pT7 + RBS)
| |
| - | {|class="hokkaidou-table-pcr-time"
| |
| - | |-
| |
| - | |Number
| |
| - | |Degree
| |
| - | |Second
| |
| - | |-
| |
| - | |1
| |
| - | |94
| |
| - | |120
| |
| - | |-
| |
| - | |2
| |
| - | |94
| |
| - | |30
| |
| - | |-
| |
| - | |3
| |
| - | |68
| |
| - | |60
| |
| - | |-
| |
| - | |4
| |
| - | |4
| |
| - | |HOLD
| |
| - | |}
| |
| - | Cycle:2~3 x 40
| |
| - | (Ag43 + dT)
| |
| - | Ag43 + dT (+ Biobrick prefis & suffix) is about 3290bp. Extension step needed >180seconds.
| |
| - | {|class="hokkaidou-table-pcr-time"
| |
| - | |-
| |
| - | |Number
| |
| - | |Degree
| |
| - | |Second
| |
| - | |-
| |
| - | |1
| |
| - | |94
| |
| - | |120
| |
| - | |-
| |
| - | |2
| |
| - | |94
| |
| - | |30
| |
| - | |-
| |
| - | |3
| |
| - | |68
| |
| - | |180
| |
| - | |-
| |
| - | |4
| |
| - | |4
| |
| - | |HOLD
| |
| - | |}
| |
| - | Cycle:2~3 x 35
| |
| - | </p>
| |
| - |
| |
| - | ===Electrophoresis results===
| |
| - | <p>
| |
| - | Electrophoresis for (pT7 + RBS) in 2% agarose gel and (Ag43 + dT) in 1% agarose gel.
| |
| - |
| |
| - |
| |
| - | pT7 + RBS on pSB1K3
| |
| - | bbp-Insert-bbs:86bp
| |
| - |
| |
| - | [[image:HokkaidoU2012 120709 pt7-rbs 75% scale.jpg|thumb|digestion result]]
| |
| - |
| |
| - |
| |
| - | Ag43 + dT on pSB1AK3
| |
| - | bbp-Insert-bbs:3290bp
| |
| - |
| |
| - | [[image:HokkaidoU2012 120709 ag43-dt-1 75% scale.jpg|thumb|digestion result]]
| |
| - |
| |
| - |
| |
| - | We couldn't confirm insert DNA were really ligated with Vector or not.
| |
| - | Next we tried confirmation of insert DNA by Electrophoresis of mini-prep products.
| |
| - | For mini-prep, we needed do liquid culture.
| |
| - | </p>
| |
| - |
| |
| - | ===Liquid culturing===
| |
| - | <p>
| |
| - | Liquid culture for mini-prep((pT7 + RBS) on pSB1K3 and (Ag43 + dT) on pSB1AK3).
| |
| - | #Prepared 1800ul LB solutions.
| |
| - | #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.
| |
| - | </p>
| |
| - | </div></div>
| |
| - |
| |
| - |
| |
| - | <div class="hokkaidou-notebook-daily">
| |
| - | ==July 10th==
| |
| - | <div>
| |
| - | ===Mini-prep===
| |
| - | <p>
| |
| - | 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).
| |
| - | #Electrophoresis (Used pre-migrated 1% agarose gels with 5ul EtBr)in 30min.
| |
| - | </p>
| |
| - |
| |
| - |
| |
| - | pT7 + RBS on pSB1K3(Total 2247bp)
| |
| - | [[image:HokkaidoU2012 120710 miniprepproduct T7+RBS.jpg|thumb|Electrophoresis resulsts]]
| |
| - |
| |
| - |
| |
| - | Ag43 + dT on pSB1AK3(Total 6444bp)
| |
| - | [[image:HokkaidoU2012 120710 miniprepproduct Ag43+dT.jpg|thumb|Electrophoresis resulsts]]
| |
| - |
| |
| - | To confirm more about insert DNA, we tried to digest these DNA with EcoRI and PstI.
| |
| - |
| |
| - | ===Digestion===
| |
| - | <p>
| |
| - | Digestion for confirmation of insert DNA. Each DNA mini-prep products were digested with E & P.
| |
| - | Digestion recipe
| |
| - | pT7-RBS
| |
| - | {|class="hokkaidou-table-digestion"
| |
| - | |-
| |
| - | |pT7-RBS
| |
| - | |1,5ul
| |
| - | |-
| |
| - | |EcoRI
| |
| - | |1ul
| |
| - | |-
| |
| - | |PstI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xH buffer
| |
| - | |2ul
| |
| - | |-
| |
| - | |DW
| |
| - | |14.5ul
| |
| - | |-
| |
| - | |Total
| |
| - | |20ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | Digestion recipe
| |
| - | Ag43-dT
| |
| - | {|class="hokkaidou-table-digestion"
| |
| - | |-
| |
| - | |Ag43-dT
| |
| - | |4ul
| |
| - | |-
| |
| - | |EcoRI
| |
| - | |1ul
| |
| - | |-
| |
| - | |PstI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xH buffer
| |
| - | |2ul
| |
| - | |-
| |
| - | |DW
| |
| - | |12ul
| |
| - | |-
| |
| - | |Total
| |
| - | |20ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | Digestioned at 37c in 2hrs.
| |
| - |
| |
| - |
| |
| - | Digestion results
| |
| - |
| |
| - | pT7+RBS
| |
| - |
| |
| - | [[image:HokkaidoU2012 120710 pt7-rbs,digestion.jpg]]
| |
| - |
| |
| - |
| |
| - | Ag43+dT
| |
| - |
| |
| - | [[image:HokkaidoU2012 120710 ag43-dt,digestion.jpg]]
| |
| - |
| |
| - |
| |
| - | Insert DNA were correct we thought. We tried digestion for 3A Assembly[pT7-RBS + Ag43-dT + pSB1C3]
| |
| - | </p>
| |
| - |
| |
| - | ===Digestion===
| |
| - | <p>
| |
| - | Digestion for 3A Assembly.
| |
| - |
| |
| - |
| |
| - | pT7-RBS
| |
| - | {|class="hokkaidou-table-digestion"
| |
| - | |-
| |
| - | |DNA
| |
| - | |17ul
| |
| - | |-
| |
| - | |EcoRI
| |
| - | |1ul
| |
| - | |-
| |
| - | |SpeI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xH buffer
| |
| - | |3ul
| |
| - | |-
| |
| - | |DW
| |
| - | |8ul
| |
| - | |-
| |
| - | |Total
| |
| - | |30ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | Ag43-dT
| |
| - | {|class="hokkaidou-table-digestion"
| |
| - | |-
| |
| - | |DNA
| |
| - | |12.5ul
| |
| - | |-
| |
| - | |XbaI
| |
| - | |1ul
| |
| - | |-
| |
| - | |PstI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xH buffer
| |
| - | |2ul
| |
| - | |-
| |
| - | |DW
| |
| - | |3.5ul
| |
| - | |-
| |
| - | |Total
| |
| - | |20ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | pSB1C3
| |
| - | {|class="hokkaidou-table-digestion"
| |
| - | |-
| |
| - | |DNA
| |
| - | |20ul
| |
| - | |-
| |
| - | |EcoRI
| |
| - | |1ul
| |
| - | |-
| |
| - | |PstI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xH buffer
| |
| - | |3ul
| |
| - | |-
| |
| - | |DW
| |
| - | |5ul
| |
| - | |-
| |
| - | |Total
| |
| - | |30ul
| |
| - | |}
| |
| - |
| |
| - | Reacted in 2hrs at 37c.
| |
| - | </p>
| |
| - | </div></div>
| |
| - |
| |
| - |
| |
| - | <div class="hokkaidou-notebook-daily">
| |
| - | ==July 11th==
| |
| - | <div>
| |
| - | ===Ligation===
| |
| - | <p>
| |
| - | Ligation of pT7-RBS + Ag43-dT + pSB1C3(3A Assembly)
| |
| - |
| |
| - |
| |
| - | Ligation recipe
| |
| - | {|class="hokkaidou-table-ligation"
| |
| - | |-
| |
| - | |pT7-RBS
| |
| - | |2ul
| |
| - | |-
| |
| - | |Ag43-dT
| |
| - | |2ul
| |
| - | |-
| |
| - | |pSB1C3
| |
| - | |3ul
| |
| - | |-
| |
| - | |Ligation Mighty Mix(TAKARA)
| |
| - | |8ul
| |
| - | |-
| |
| - | |Total
| |
| - | |16ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | Ligation reaction time was written below.
| |
| - |
| |
| - | {|class="hokkaidou-table-ligation"
| |
| - | |-
| |
| - | |Degree
| |
| - | |Minute
| |
| - | |-
| |
| - | |16
| |
| - | |30
| |
| - | |-
| |
| - | |65
| |
| - | |10
| |
| - | |-
| |
| - | |4
| |
| - | |Hold
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | Electrophoresis result
| |
| - |
| |
| - | [[image:HokkaidoU2012 120711 pt7-rbs-ag43-dtjpg.jpg]]
| |
| - |
| |
| - |
| |
| - | ===Transformation===
| |
| - | Transformation for pT7-RBS + Ag43-dT + pSB1C3 into BL21(DE3)(E.coli which have T7 polymerase coadin site in their genomic DNA).
| |
| - | #Added DNA soltions (Ligation products) 1ul to BL21(DE3) compitent cell.
| |
| - | #Stood on ice in 30min.
| |
| - | #Heatshock for 1min at 42c.
| |
| - | #Added 200ul of LB to transformed BL21(DE3) solution.
| |
| - | #Pre-cultivate in 2hrs
| |
| - | #Splead 200ul of LB&BL21(DE3) solution supernant to LBC.
| |
| - | #50ul of LB&BL21(DE3) solution were added to 200ul LB solution then spread 200ul to LBC plate.
| |
| - | #Cultivated.
| |
| - | </p>
| |
| - | </div></div>
| |
| - |
| |
| - | <div class="hokkaidou-notebook-daily">
| |
| - | ==July 12th==
| |
| - | <div>
| |
| - | ===Colony PCR===
| |
| - | <p>
| |
| - | 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
| |
| - | |4ul
| |
| - | |-
| |
| - | |KapaTaq ready mix
| |
| - | |5ul
| |
| - | |-
| |
| - | |BioBrick prefix forward primer
| |
| - | |0.5ul
| |
| - | |-
| |
| - | |BioBrick suffix reverse primer
| |
| - | |0.5ul
| |
| - | |-
| |
| - | |Total
| |
| - | |10ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | '''PCR recipe'''
| |
| - |
| |
| - | (pT7 + RBS)
| |
| - | {|class="hokkaidou-table-pcr-time"
| |
| - | |-
| |
| - | |Number
| |
| - | |Degree
| |
| - | |Second
| |
| - | |-
| |
| - | |1
| |
| - | |94
| |
| - | |120
| |
| - | |-
| |
| - | |2
| |
| - | |94
| |
| - | |30
| |
| - | |-
| |
| - | |3
| |
| - | |68
| |
| - | |90
| |
| - | |-
| |
| - | |4
| |
| - | |4
| |
| - | |HOLD
| |
| - | |}
| |
| - | Cycle:2~3 x 40
| |
| - | </p>
| |
| - |
| |
| - | ===Liquid culturing===
| |
| - | <p>
| |
| - | 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.
| |
| - | </p>
| |
| - | </div></div>
| |
| - |
| |
| - | <div class="hokkaidou-notebook-daily">
| |
| - | ==July 13th==
| |
| - | <div>
| |
| - | ===Mini-prep===
| |
| - | <p>
| |
| - | Mini-prep for colony No.1~8
| |
| - | We used mini-prep kit FastGene Plasmid Mini Kit (Nippon Genetics).
| |
| - |
| |
| - |
| |
| - | Mini-prep result
| |
| - |
| |
| - | [[image:HokkaidoU2012 120713 pT7-RBS-Ag43-dT on pSB1C3 mini-prep.jpg]]
| |
| - | </p>
| |
| - |
| |
| - | ===Digestion===
| |
| - | <p>
| |
| - | Digestion to confirm how DNA fragments ligated.
| |
| - |
| |
| - |
| |
| - | Digestion Recipe
| |
| - | {|class="hokkaidou-table-digestion"
| |
| - | |-
| |
| - | |DNA
| |
| - | |4ul
| |
| - | |-
| |
| - | |EcoRI
| |
| - | |0.5ul
| |
| - | |-
| |
| - | |PstI
| |
| - | |0.5ul
| |
| - | |-
| |
| - | |10xH buffer
| |
| - | |2ul
| |
| - | |-
| |
| - | |DW
| |
| - | |13ul
| |
| - | |-
| |
| - | |Total
| |
| - | |20ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | Digestion result
| |
| - |
| |
| - | [[image:HokkaidoU2012 120714 pT7-RBS-Ag43-dT on pSB1C3 digeation EP .jpg]]
| |
| - | </p>
| |
| - | </div></div>
| |
| - |
| |
| - | <div class="hokkaidou-notebook-daily">
| |
| - | ==July 14th==
| |
| - | <div>
| |
| - | Blue Screen!!!!
| |
| - | Blue Screen!!!!
| |
| - | Blue Screen!!!!
| |
| - | 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!!!!
| |
| - | Blue Screen!!!!
| |
| - | Blue Screen!!!!
| |
| - | Blue Screen!!!!
| |
| - | Blue Screen!!!!
| |
| - | Blue Screen!!!!
| |
| - | Blue Screen!!!!
| |
| - | Blue Screen!!!!
| |
| - | Blue Screen!!!!
| |
| - | Blue Screen!!!!
| |
| - | Blue Screen!!!!
| |
| - | Blue Screen!!!!
| |
| - | Blue Screen!!!!
| |
| - | Blue Screen!!!!
| |
| - | Blue Screen!!!!
| |
| - | Blue Screen!!!!
| |
| - | Blue Screen!!!!
| |
| - | Blue Screen!!!!
| |
| - | Blue Screen!!!!
| |
| - | Blue Screen!!!!
| |
| - |
| |
| - |
| |
| - | ===Ligation===
| |
| - | <p>
| |
| - | 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
| |
| - | 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]]
| |
| - | </p>
| |
| - |
| |
| - | ===Transformation===
| |
| - | <p>
| |
| - | 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
| |
| - | #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 in 21hrs.
| |
| - |
| |
| - |
| |
| - |
| |
| - | 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]]
| |
| - | </p>
| |
| - |
| |
| - | ===Liquid culture===
| |
| - | <p>
| |
| - | Liquid culture for Ag43(BBa_K346007)
| |
| - | #Picked up one colony from single colony isolated plate.
| |
| - | #Dipped into LBC.
| |
| - | #cultivated.
| |
| - | </p>
| |
| - | </div></div>
| |
| - |
| |
| - | <div class="hokkaidou-notebook-daily">
| |
| - | ==July 15th==
| |
| - | <div>
| |
| - | ===Gel extraction===
| |
| - | <p>
| |
| - | We used Gel Extraction Kit (FastGene Gel/PCR extraction kit:NipponGenetics) to purify digestion products (see 14th).
| |
| - | </p>
| |
| - |
| |
| - | ===Digestion===
| |
| - | <p>
| |
| - | Digestion to confirm what kind of restriction enzyme cutting sites these DNA (colony 1 and 6) have.
| |
| - |
| |
| - | EcoRI
| |
| - | {|class="hokkaidou-table-digestion"
| |
| - | |-
| |
| - | |DNA solution
| |
| - | |6ul
| |
| - | |-
| |
| - | |EcoRI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xH buffer
| |
| - | |1ul
| |
| - | |-
| |
| - | |DW
| |
| - | |2ul
| |
| - | |-
| |
| - | |Total
| |
| - | |10ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | XbaI
| |
| - | {|class="hokkaidou-table-digestion"
| |
| - | |-
| |
| - | |DNA solution
| |
| - | |6ul
| |
| - | |-
| |
| - | |XbaI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xM buffer
| |
| - | |1ul
| |
| - | |-
| |
| - | |BSA
| |
| - | |1ul
| |
| - | |-
| |
| - | |DW
| |
| - | |1ul
| |
| - | |-
| |
| - | |Total
| |
| - | |10ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | PstI
| |
| - | {|class="hokkaidou-table-digestion"
| |
| - | |-
| |
| - | |DNA solution
| |
| - | |6ul
| |
| - | |-
| |
| - | |PstI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xH buffer
| |
| - | |1ul
| |
| - | |-
| |
| - | |DW
| |
| - | |2ul
| |
| - | |-
| |
| - | |Total
| |
| - | |10ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - |
| |
| - | SpeI
| |
| - | {|class="hokkaidou-table-digestion"
| |
| - | |-
| |
| - | |DNA solution
| |
| - | |6ul
| |
| - | |-
| |
| - | |SpeI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xM buffer
| |
| - | |1ul
| |
| - | |-
| |
| - | |DW
| |
| - | |2ul
| |
| - | |-
| |
| - | |Total
| |
| - | |10ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | EcoRI + PstI
| |
| - | {|class="hokkaidou-table-digestion"
| |
| - | |-
| |
| - | |DNA solution
| |
| - | |6ul
| |
| - | |-
| |
| - | |EcoRI
| |
| - | |1ul
| |
| - | |-
| |
| - | |PstI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xH buffer
| |
| - | |1ul
| |
| - | |-
| |
| - | |DW
| |
| - | |11ul
| |
| - | |-
| |
| - | |Total
| |
| - | |20ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | XbaI + SpeI
| |
| - | {|class="hokkaidou-table-digestion"
| |
| - | |-
| |
| - | |DNA solution
| |
| - | |6ul
| |
| - | |-
| |
| - | |XbaI
| |
| - | |1ul
| |
| - | |-
| |
| - | |SpeI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xM buffer
| |
| - | |1ul
| |
| - | |-
| |
| - | |DW
| |
| - | |11ul
| |
| - | |-
| |
| - | |Total
| |
| - | |20ul
| |
| - | |}
| |
| - | </p>
| |
| - |
| |
| - | ===Ethanol precipitation===
| |
| - | <p>
| |
| - | Ethanol precipitation for digestion products.
| |
| - | #Added 2ul of NaoAc, 1.5ul of glycogen and 50ul of 100% ethanol.
| |
| - | #Centrifuged in 15000rpm, 10min at 4C.
| |
| - | #Remove supernatant and added 220ul of 70% ethanol.
| |
| - | #Centrifuged in 15000rpm, 5min at 4C.
| |
| - | #Remove supernatant and air drying in room temperature then added 5ul of DW.
| |
| - | </p>
| |
| - |
| |
| - | ===Electrophoresis===
| |
| - | <p>
| |
| - | Electrophoresis for digest-ethanol precipitation products.
| |
| - | #added 5ul of EtBr.
| |
| - | #Migrated in 30min.
| |
| - |
| |
| - |
| |
| - | '''Digestion result'''
| |
| - |
| |
| - | Digestion results for pT7-RBS-RFP-dT on pSB1C3
| |
| - | (once digested with EcoRI and PstI)
| |
| - |
| |
| - | [[image:HokkaidoU2012 120715 pT7-rbs-ag43-dt digestion(X,S,P,E,EP,XS).jpg]]
| |
| - | </p>
| |
| - |
| |
| - | ===Single colony isolation===
| |
| - | <p>
| |
| - | Single colony isolation for Transformation products synthesized yesterday.
| |
| - | #one colony picked up from cultivated LBC and LBT plate.
| |
| - | #Spread on LBC and LBT.
| |
| - | #Cultivated.
| |
| - | </p>
| |
| - |
| |
| - | ===Digestion===
| |
| - | <p>
| |
| - | Digestion of Ag43(with S,P), dT(With X,P) and pT7-RBS(With E).
| |
| - |
| |
| - |
| |
| - | Ag43
| |
| - | {|class="hokkaidou-table-digestion"
| |
| - | |-
| |
| - | |DNA solution
| |
| - | |9ul
| |
| - | |-
| |
| - | |SpeI
| |
| - | |1ul
| |
| - | |-
| |
| - | |PstI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xH buffer
| |
| - | |2ul
| |
| - | |-
| |
| - | |DW
| |
| - | |7ul
| |
| - | |-
| |
| - | |Total
| |
| - | |20ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | dT
| |
| - | {|class="hokkaidou-table-digestion"
| |
| - | |-
| |
| - | |DNA solution
| |
| - | |1.1ul
| |
| - | |-
| |
| - | |XbaI
| |
| - | |1ul
| |
| - | |-
| |
| - | |PstI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xM buffer
| |
| - | |2ul
| |
| - | |-
| |
| - | |DW
| |
| - | |14.9ul
| |
| - | |-
| |
| - | |Total
| |
| - | |20ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | pT7-RBS
| |
| - | {|class="hokkaidou-table-digestion"
| |
| - | |-
| |
| - | |DNA solution
| |
| - | |6ul
| |
| - | |-
| |
| - | |EcoRI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xH buffer
| |
| - | |2ul
| |
| - | |-
| |
| - | |DW
| |
| - | |11ul
| |
| - | |-
| |
| - | |Total
| |
| - | |20ul
| |
| - | |}
| |
| - | </p>
| |
| - | </div></div>
| |
| - | <!-- DO NOT EDIT UNDER THIS LINE @iTakeshi -->
| |
| - | </div>
| |
| - | <br style="line-height: 0; clear: both;" />
| |
| - | {{Team:HokkaidoU_Japan/footer}}
| |
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