|
|
| (17 intermediate revisions not shown) |
| Line 1: |
Line 1: |
| - | ==2nd==
| |
| - | <div class="hokkaidou-notebook-daily">
| |
| - | On your mark...
| |
| - | </div>
| |
| | | | |
| - | ==3rd==
| |
| - | <div class="hokkaidou-notebook-daily">
| |
| - | Get set...
| |
| - | </div>
| |
| - |
| |
| - | ==4th==
| |
| - | <div class="hokkaidou-notebook-daily">
| |
| - | ;Transformation
| |
| - | Transformation of BBa_B0015(dT), B0034(RBS), I179005(pT7), K346007(Ag43), and K542009(pLacI-RBS-Ag43) in DH5α
| |
| - | #Added each DNA solutions (1ul) into DH5α comeptent cell and standed on ice in 30 min.
| |
| - | #Cultivated on LBA(dt,RBS,T7) and LBC(Ag43, pLacI-RBS-Ag43). E.coli cultivate in LBC was pre-cultivated in 2 hrs. pT7 was 21hrs and Others were 20hrs cultivated
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | ==5th==
| |
| - | <div class="hokkaidou-notebook-daily">
| |
| - |
| |
| - | ;Transformation
| |
| - | K346007(Ag43) was failed to cultivate on LBC plate.
| |
| - | Transformation of K346007(Ag43) in DH5α.
| |
| - | #Add DNA solution(1ul) into DH5α comeptent cell and stand on ice in 30 min.
| |
| - | #Pre-cultivated in 2hrs.
| |
| - | #Cultivated on LBC in 21hrs.
| |
| - |
| |
| - | ;Single colony isolation
| |
| - | Single colony isolation of BBa_B0015, B0034, I179005 and K542009.
| |
| - | #Picked up one colony.
| |
| - | #Cultivation on LBA(dt,RBS,T7) and LBC(pLacI-RBS-Ag43) in 14hrs30mins
| |
| - | BBa_K542009 was Ag43 only part! And the part didn't have Biobrick suffix.
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | ==6th==
| |
| - | <div class="hokkaidou-notebook-daily">
| |
| - | ;Liquid culture
| |
| - | Liquid culture in LBA(dT,RBS,pT7) and LBC(pLacI-RBS-Ag43)
| |
| - | #Picked up two colonies from each plates.
| |
| - | #One colony was dipped in 1ml LB (A or C), and other colony was dipped in 2ml LB(A or C). 1ml is for glycerol stocks and 2ml is for mini-prep.
| |
| - | #16hrs Cultivation
| |
| - | <br />
| |
| - | ;Single colony isolation
| |
| - | #Single colony isolation of K346007(Ag43).
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | ==7th==
| |
| - | <div class="hokkaidou-notebook-daily">
| |
| - |
| |
| - | ;Liquid culture
| |
| - | Liquid culture in LBC(Ag43).
| |
| - | #Picked up two colonies from each plates.
| |
| - | #Both of colonies were dipped in 2ml LBC, and then we cultivate them in 38℃.<br>
| |
| - | However, one of them cultivated only 8 hours. It's for glycerol stock.
| |
| - | <br />
| |
| - |
| |
| - |
| |
| - | '''3A assembly!'''<br>
| |
| - | Assembled pT7, RBS and pSB1C3 by 3A assembly.
| |
| - | This 3A assembly is our first try!
| |
| - |
| |
| - | ;mini-prep
| |
| - | #mini-prep of dT,RBS,pT7 and pLacI-RBS-Ag43.
| |
| - | #Elution in 50ul buffer
| |
| - | <br />
| |
| - |
| |
| - | ;Glycerol stock
| |
| - | Made glycerol stocks of dT,RBS,pT7 and pLacI-RBS-Ag43.
| |
| - | #Parts written above were cultivated in LBA(dT,RBS,pT7) and LBC(pLacI-RBS-Ag43) 1ml in 16hrs 30min.
| |
| - | #Add glycerol and Freeze at -80C
| |
| - | <br />
| |
| - |
| |
| - | [[Image:120707_I719005_B0034_B0015_K542009_mini-prep_umeuchi.jpg]]
| |
| - | ;Electrophoresis
| |
| - | Electrophoresis to predict concentration of mini-prep products(dT,RBS,pT7 and pLacI-RBS-Ag43).
| |
| - | #Used 1% agarose gel.
| |
| - | #Pre-migration.
| |
| - | #Migrated 1.2ul of DNA solutions (1ul is mini-prep products and 0.2ul is Loading Dye) in 35min.
| |
| - | #Took a photograph of 1% agarose gel that finished electrophoresis.
| |
| - | <br />
| |
| - |
| |
| - | ;Digestion
| |
| - | <br />
| |
| - | Digestion of I719005, B0034 and pSB1K3
| |
| - |
| |
| - | Digestion recipe
| |
| - |
| |
| - | All parts were reacted in 30ul solution.
| |
| - | *I719005(40ng/ul)
| |
| - | {|class="hokkaidou-table-digestion"
| |
| - | |-
| |
| - | |DNA solution
| |
| - | |12.5ul
| |
| - | |-
| |
| - | |EcoRI
| |
| - | |1ul
| |
| - | |-
| |
| - | |SpeI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xH Buffer
| |
| - | |3ul
| |
| - | |-
| |
| - | |DW
| |
| - | |12.5ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | *B0034(40ng/ul)
| |
| - | {|class="hokkaidou-table-digestion"
| |
| - | |-
| |
| - | |DNA solution
| |
| - | |12.5ul
| |
| - | |-
| |
| - | |XbaI
| |
| - | |1ul
| |
| - | |-
| |
| - | |PstI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xM Buffer
| |
| - | |3ul
| |
| - | |-
| |
| - | |DW
| |
| - | |12.5ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | *pSB1K3(25ng/ul)
| |
| - | {|class="hokkaidou-table-digestion"
| |
| - | |-
| |
| - | |DNA solution
| |
| - | |12ul
| |
| - | |-
| |
| - | |EcoRI
| |
| - | |1ul
| |
| - | |-
| |
| - | |PstI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xH Buffer
| |
| - | |3ul
| |
| - | |-
| |
| - | |DW
| |
| - | |13ul
| |
| - | |}
| |
| - | <br />
| |
| - | ;Ethanol precipitation
| |
| - | For rising concentration of DNA solution which use for Ligation and removing restriction enzyme.
| |
| - | #Added 3ul of NaoAc, 1.5ul of glycogen and 75ul of 100% ethanol.
| |
| - | #Centrifuged in 14000rpm, 30min at 4C.
| |
| - | #Remove supernatant and added 220ul of 70% ethanol.
| |
| - | #Centrifuged in 15000rpm, 15min at 4C.
| |
| - | #Remove supernatant and air drying in room temperature then added 10ul of DW.
| |
| - | <br />
| |
| - | ;Ligation
| |
| - | All DNA solutions were digested.
| |
| - | 3A assembly protocol required Ligation reaction should be in total 25ul solution.
| |
| - | {|class="hokkaidou-table-ligation"
| |
| - | |-
| |
| - | |Ligation Mighty Mix
| |
| - | |12.5ul
| |
| - | |-
| |
| - | |pT7
| |
| - | |2ul
| |
| - | |-
| |
| - | |RBS
| |
| - | |2ul
| |
| - | |-
| |
| - | |pSB1K3
| |
| - | |2ul
| |
| - | |-
| |
| - | |DW
| |
| - | |6.5ul
| |
| - | |-
| |
| - | |Total
| |
| - | |25ul
| |
| - | |}
| |
| - |
| |
| - | Ligation reaction recipe was written below.
| |
| - |
| |
| - | {|class="hokkaidou-table-pcr-time"
| |
| - | |-
| |
| - | |Degree
| |
| - | |Minute
| |
| - | |-
| |
| - | |16
| |
| - | |30
| |
| - | |-
| |
| - | |65
| |
| - | |10
| |
| - | |-
| |
| - | |4
| |
| - | |Hold
| |
| - | |}
| |
| - | <br />
| |
| - | ligation was finished.
| |
| - | But now pm10:00. 2.5hrs needs for doing transformation. Transformation would finish at am:0:30.
| |
| - |
| |
| - | Withdraw!!!!
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | ==8th==
| |
| - | <div class="hokkaidou-notebook-daily">
| |
| - | *(pT7 + RBS)
| |
| - | ;Transformation
| |
| - | Transformation for pT7+RBS+pSB1K3
| |
| - | #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 LBK.
| |
| - | #Cultivated
| |
| - | <br />
| |
| - |
| |
| - | *K346007(Ag43)
| |
| - | ;mini-prep
| |
| - | mini-prep for Liquid culture product of K346007(Ag43)
| |
| - | #Used FastGene Plasmid Mini Kit(Nippon Genetics)
| |
| - | #Elutioned in 50ul
| |
| - | #First we eluted in colection tube. then moved in Eppendorf tube.
| |
| - |
| |
| - |
| |
| - | ;Erectrophoresis
| |
| - | Erectrophoresis for mini-prep product(Ag43).
| |
| - | #Prepared 1% Agalose gel and added EtBr then pre-migration in 30min.
| |
| - | #1ul 1kb ladder, 1.2ul mini-prep product(1ul is DNA solution and 0.2ul is loading dye) added then migtrated
| |
| - |
| |
| - | mini-prep result (With ligation result of pT7+RBS+pSB1K3)
| |
| - |
| |
| - | [[image:HokkaidoU2012 120708 K346007 miniprep and pt7 rbs.jpg]]
| |
| - |
| |
| - | ;Glycerol stock
| |
| - | Made glycerol stock of K346007 (Ag43).
| |
| - | #Parts written above were cultivated in LBC.
| |
| - | #Added glycerol and Freezed at -80C
| |
| - |
| |
| - |
| |
| - | *(Ag43 + dT)
| |
| - | Assembling K346007(Ag43) + B0015(dT) with 2-piece assembly(Biobrisk standard assembly)
| |
| - |
| |
| - |
| |
| - | ;Digestion
| |
| - | Digested Ag43 and dT in solution by recipes Written below.
| |
| - | Insert DNA required too much weight and volume(volume was calculated from concentration of DNA mini-prep
| |
| - | product)from our calculation. There are no insurance of succession of digestion.
| |
| - |
| |
| - | *Ag43(Insert)
| |
| - | 5190bp(Ag43 + pSB1C3)
| |
| - | {|class="hokkaidou-table-digestion"
| |
| - | |-
| |
| - | |DNA solution
| |
| - | |48ul
| |
| - | |-
| |
| - | |EcoRI
| |
| - | |1ul
| |
| - | |-
| |
| - | |SpeI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xH buffer
| |
| - | |6ul
| |
| - | |-
| |
| - | DW
| |
| - | |4ul
| |
| - | |-
| |
| - | |Total
| |
| - | |60ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | *dT(Vector)
| |
| - | 3318bp(Ag43 + pSB1AK3)
| |
| - | {|class="hokkaidou-table-digestion"
| |
| - | |-
| |
| - | |DNA solution
| |
| - | |8ul
| |
| - | |-
| |
| - | |EcoRI
| |
| - | |1ul
| |
| - | |-
| |
| - | |XbaI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xM buffer
| |
| - | |2ul
| |
| - | |-
| |
| - | |DW
| |
| - | |8ul
| |
| - | |-
| |
| - | |Total
| |
| - | |20ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | Digestion result image
| |
| - |
| |
| - |
| |
| - | [[image:HokkaidoU2012 120708 dt K346007 digestion umeuchi.jpg]]
| |
| - |
| |
| - |
| |
| - | K346007(Ag43) was 5190bp before digestion (Biobrick prefix + Ag43 + Biobrick suffix + pSB1C3).
| |
| - | After digestion, Ag43 and pSB1C3 was separated and became fragments about 3120bp(Ag43) and 2070bp(pSB1C3).
| |
| - | Gel image above shows there are two fragments, one fragment is about 2000bp other fragment is 3000bp.
| |
| - | Digestion would be succeeded.
| |
| - | About Vector(Boo15:dT), the DNA was circular so DNA migrated more far than Linear DNA. d+ (Circular DNA) migrated little more far than d- (Linear DNA). so we think digestion was succeeded.
| |
| - |
| |
| - |
| |
| - | ;Ethanol precipitation
| |
| - | Ethanol precipitation for gel extraction products(K346007(Ag43) and B0015(dT))
| |
| - | #Added 5ul of NaoAc , 1.5ul of glycogen and 125ul of 100% ethanol to 50ul DNA solutions.
| |
| - | #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 10ul of DW.
| |
| - |
| |
| - | ;Ligation
| |
| - | All DNA solutions were digested.
| |
| - | Used Ligation Mighty Mix(TakaraBio)
| |
| - |
| |
| - | {|class="hokkaidou-table-ligation"
| |
| - | |-
| |
| - | |Ligation Mighty Mix
| |
| - | |5ul
| |
| - | |-
| |
| - | |Insert: Ag43
| |
| - | |2ul
| |
| - | |-
| |
| - | |Vector: dT
| |
| - | |2ul
| |
| - | |-
| |
| - | |DW
| |
| - | |1ul
| |
| - | |-
| |
| - | |Total
| |
| - | |10ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | Ligation reaction recipe was written below.
| |
| - |
| |
| - | {|class="hokkaidou-table-ligation"
| |
| - | |-
| |
| - | |Degree
| |
| - | |Minute
| |
| - | |-
| |
| - | |16
| |
| - | |30
| |
| - | |-
| |
| - | |65
| |
| - | |10
| |
| - | |-
| |
| - | |4
| |
| - | |Hold
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | ;Electrophoresis
| |
| - | Confirmation of succession of ligation.
| |
| - | #Prepared 1% Agalose gel and added EtBr then pre-migration in 30min.
| |
| - | #Added 1kb ladder, Ligation product(1ul) and digestion products (control:each solutions 1ul).
| |
| - | #Migtrated in 30min.
| |
| - |
| |
| - | Electrophoresis results
| |
| - |
| |
| - |
| |
| - | [[image:HokkaidoU2012 120708 K346007-dT ligation.jpg]]
| |
| - |
| |
| - |
| |
| - | ;Transformation
| |
| - | Transformation for K346007(Ag43)+B0015(dT) on pSB1AK3.
| |
| - | #Added DNA soltions (Ligation products) 1ul to DH5α compitent cell.
| |
| - | #Stood on ice in 30min.
| |
| - | #Added 600ul of LB to transformed DH5α solution.
| |
| - | #From 700 solution(100ul is DH5α and 600ul is LB), 100ul add to 900ul of LB(x10 solution)
| |
| - | #Spread 300ul from 600(700-100)ul and 1000ul of LB&DH5α solution to each LBA plates.
| |
| - | #Cultivated.
| |
| - | </div>
| |
"
