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| - | *4th
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| | | | |
| - | ;Transformation
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| - | Transformation of BBa_B0015(dT), B0034(RBS), I179005(pT7), K346007(Ag43), and K542009(pLacI-RBS-Ag43) in DH5α
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| - | #Added each DNA solutions (1ul) into DH5α comeptent cell and standed on ice in 30 min.
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| - | #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
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| - | <br>
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| - |
| |
| - | ----
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| - | *5th
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| - |
| |
| - | ;Transformation
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| - | K346007(Ag43) was failed to cultivate on LBC plate.
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| - | Transformation of K346007(Ag43) in DH5α.
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| - | #Add DNA solution(1ul) into DH5α comeptent cell and stand on ice in 30 min.
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| - | #Pre-cultivated in 2hrs.
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| - | #Cultivated on LBC in 21hrs.
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| - |
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| - | ;Single colony isolation
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| - | Single colony isolation of BBa_B0015, B0034, I179005 and K542009.
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| - | #Picked up one colony.
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| - | #Cultivation on LBA(dt,RBS,T7) and LBC(pLacI-RBS-Ag43) in 14hrs30mins
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| - | BBa_K542009 was Ag43 only part! And the part didn't have Biobrick suffix.
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| - |
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| - |
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| - | ----
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| - | *6th
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| - |
| |
| - | ;Liquid culture
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| - | Liquid culture in LBA(dT,RBS,pT7) and LBC(pLacI-RBS-Ag43)
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| - | #Picked up two colonies from each plates.
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| - | #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.
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| - | #16hrs Cultivation
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| - | <br>
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| - | ;Single colony isolation
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| - | #Single colony isolation of K346007(Ag43).
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| - | <br>
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| - |
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| - | ----
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| - | *7th
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| - |
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| - |
| |
| - | ;Liquid culture
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| - | Liquid culture in LBC(Ag43).
| |
| - | #Picked up two colonies from each plates.
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| - | #Both of colonies were dipped in 2ml LBC, and then we cultivate them in 38℃.<br>
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| - | However, one of them cultivated only 8 hours. It's for glycerol stock.
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| - | <br>
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| - |
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| - |
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| - | '''3A assembly!'''<br>
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| - | Assembled pT7, RBS and pSB1C3 by 3A assembly.
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| - | This 3A assembly is our first try!
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| - |
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| - | ;mini-prep
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| - | #mini-prep of dT,RBS,pT7 and pLacI-RBS-Ag43.
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| - | #Elution in 50ul buffer
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| - | <br>
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| - |
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| - | ;Glycerol stock
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| - | Made glycerol stocks of dT,RBS,pT7 and pLacI-RBS-Ag43.
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| - | #Parts written above were cultivated in LBA(dT,RBS,pT7) and LBC(pLacI-RBS-Ag43) 1ml in 16hrs 30min.
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| - | #Add glycerol and Freeze at -80C
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| - | <br>
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| - |
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| - | [[Image:120707_I719005_B0034_B0015_K542009_mini-prep_umeuchi.jpg]]
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| - | ;Electrophoresis
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| - | Electrophoresis to predict concentration of mini-prep products(dT,RBS,pT7 and pLacI-RBS-Ag43).
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| - | #Used 1% agarose gel.
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| - | #Pre-migration.
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| - | #Migrated 1.2ul of DNA solutions (1ul is mini-prep products and 0.2ul is Loading Dye) in 35min.
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| - | #Took a photograph of 1% agarose gel that finished electrophoresis.
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| - | <br>
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| - |
| |
| - | ;Digestion
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| - | <br>
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| - | Digestion of I719005, B0034 and pSB1K3
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| - |
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| - | Digestion recipe
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| - |
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| - | All parts were reacted in 30ul solution.
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| - | *I719005(40ng/ul)
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| - | {|
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| - | |DNA solution
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| - | |12.5ul
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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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| - | |12.5ul
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| - | |}
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| - |
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| - |
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| - | *B0034(40ng/ul)
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| - | {|
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| - | |DNA solution
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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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| - | |10xM Buffer
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| - | |3ul
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| - | |-
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| - | |DW
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| - | |12.5ul
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| - | |}
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| - |
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| - |
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| - | *pSB1K3(25ng/ul)
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| - | {|
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| - | |DNA solution
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| - | |12ul
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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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| - | |13ul
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| - | |}
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| - | <br>
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| - | ;Ethanol precipitation
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| - | For rising concentration of DNA solution which use for Ligation and removing restriction enzyme.
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| - | #Added 3ul of NaoAc, 1.5ul of glycogen and 75ul of 100% ethanol.
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| - | #Centrifuged in 14000rpm, 30min at 4C.
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| - | #Remove supernatant and added 220ul of 70% ethanol.
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| - | #Centrifuged in 15000rpm, 15min at 4C.
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| - | #Remove supernatant and air drying in room temperature then added 10ul of DW.
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| - | <br>
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| - | ;Ligation
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| - | All DNA solutions were digested.
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| - | 3A assembly protocol required Ligation reaction should be in total 25ul solution.
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| - | {|
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| - | |Ligation Mighty Mix
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| - | |12.5ul
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| - | |-
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| - | |pT7
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| - | |2ul
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| - | |-
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| - | |RBS
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| - | |2ul
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| - | |-
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| - | |pSB1K3
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| - | |2ul
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| - | |-
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| - | |DW
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| - | |6.5ul
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| - | |-
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| - | |――――――――――
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| - | |-
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| - | |Total
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| - | |25ul
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| - | |}
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| - |
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| - | Ligation reaction recipe was written below.
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| - |
| |
| - | {|
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| - | |Degree
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| - | |Minute
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| - | |-
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| - | |16
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| - | |30
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| - | |-
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| - | |65
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| - | |10
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| - | |-
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| - | |4
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| - | |Hold
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| - | |}
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| - | <br>
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| - | ligation was finished.
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| - | But now pm10:00. 2.5hrs needs for doing transformation. Transformation would finish at am:0:30.
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| - |
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| - | Withdraw!!!!
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| - |
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| - | ----
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| - | *8th
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| - |
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| - | *(pT7 + RBS)
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| - | ;Transformation
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| - | Transformation for pT7+RBS+pSB1K3
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| - | #Added DNA soltions (Ligation products) 1ul to DH5α compitent cell.
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| - | #Stood on ice in 30min.
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| - | #Added 600ul of LB to transformed DH5α solution.
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| - | #Pre-cultivate in 2hrs
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| - | #Splead 300ul of LB&DH5α solution to LBK.
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| - | #Cultivated
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| - | <br>
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| - |
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| - | *K346007(Ag43)
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| - | ;mini-prep
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| - | mini-prep for Liquid culture product of K346007(Ag43)
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| - | #Used FastGene Plasmid Mini Kit(Nippon Genetics)
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| - | #Elutioned in 50ul
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| - | #First we eluted in colection tube. then moved in Eppendorf tube.
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| - |
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| - |
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| - | ;Erectrophoresis
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| - | Erectrophoresis for mini-prep product(Ag43).
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| - | #Prepared 1% Agalose gel and added EtBr then pre-migration in 30min.
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| - | #1ul 1kb ladder, 1.2ul mini-prep product(1ul is DNA solution and 0.2ul is loading dye) added then migtrated
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| - |
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| - | mini-prep result (With ligation result of pT7+RBS+pSB1K3)
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| - |
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| - | [[image:HokkaidoU2012 120708 K346007 miniprep and pt7 rbs.jpg]]
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| - |
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| - | ;Glycerol stock
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| - | Made glycerol stock of K346007 (Ag43).
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| - | #Parts written above were cultivated in LBC.
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| - | #Added glycerol and Freezed at -80C
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| - |
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| - |
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| - | *(Ag43 + dT)
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| - | Assembling K346007(Ag43) + B0015(dT) with 2-piece assembly(Biobrisk standard assembly)
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| - |
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| - |
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| - | ;Digestion
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| - | Digested Ag43 and dT in solution by recipes Written below.
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| - | Insert DNA required too much weight and volume(volume was calculated from concentration of DNA mini-prep
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| - | product)from our calculation. There are no insurance of succession of digestion.
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| - |
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| - | *Ag43(Insert)
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| - | 5190bp(Ag43 + pSB1C3)
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| - | {|
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| - | |DNA solution
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| - | |48ul
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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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| - | |6ul
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| - | |-
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| - | DW
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| - | |4ul
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| - | |-
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| - | |――――――――――――
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| - | |-
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| - | |Total
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| - | |60ul
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| - | |}
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| - |
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| - |
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| - | *dT(Vector)
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| - | 3318bp(Ag43 + pSB1AK3)
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| - | {|
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| - | |DNA solution
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| - | |8ul
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| - | |-
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| - | |EcoRI
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| - | |1ul
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| - | |-
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| - | |XbaI
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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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| - | |8ul
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| - | |-
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| - | |――――――――――――
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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 result image
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| - |
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| - |
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| - | [[image:HokkaidoU2012 120708 dt K346007 digestion umeuchi.jpg]]
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| - |
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| - |
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| - | K346007(Ag43) was 5190bp before digestion (Biobrick prefix + Ag43 + Biobrick suffix + pSB1C3).
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| - | After digestion, Ag43 and pSB1C3 was separated and became fragments about 3120bp(Ag43) and 2070bp(pSB1C3).
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| - | Gel image above shows there are two fragments, one fragment is about 2000bp other fragment is 3000bp.
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| - | Digestion would be succeeded.
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| - | 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.
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| - |
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| - |
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| - | ;Ethanol precipitation
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| - | Ethanol precipitation for gel extraction products(K346007(Ag43) and B0015(dT))
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| - | #Added 5ul of NaoAc , 1.5ul of glycogen and 125ul of 100% ethanol to 50ul DNA solutions.
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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, 5min at 4C.
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| - | #Remove supernatant and air drying in room temperature then added 10ul of DW.
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| - |
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| - | ;Ligation
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| - | All DNA solutions were digested.
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| - | Used Ligation Mighty Mix(TakaraBio)
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| - |
| |
| - | {|
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| - | |Ligation Mighty Mix
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| - | |5ul
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| - | |-
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| - | |Insert: Ag43
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| - | |2ul
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| - | |-
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| - | |Vector: dT
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| - | |2ul
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| - | |-
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| - | |DW
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| - | |1ul
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| - | |-
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| - | |――――――――――
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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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| - | Ligation reaction recipe was written below.
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| - |
| |
| - | {|
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| - | |Degree
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| - | |Minute
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| - | |-
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| - | |16
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| - | |30
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| - | |-
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| - | |65
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| - | |10
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| - | |-
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| - | |4
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| - | |Hold
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| - | |}
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| - |
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| - |
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| - | ;Electrophoresis
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| - | Confirmation of succession of ligation.
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| - | #Prepared 1% Agalose gel and added EtBr then pre-migration in 30min.
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| - | #Added 1kb ladder, Ligation product(1ul) and digestion products (control:each solutions 1ul).
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| - | #Migtrated in 30min.
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| - |
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| - | Electrophoresis results
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| - |
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| - |
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| - | [[image:HokkaidoU2012 120708 K346007-dT ligation.jpg]]
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| - |
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| - |
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| - | ;Transformation
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| - | Transformation for K346007(Ag43)+B0015(dT) on pSB1AK3.
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| - | #Added DNA soltions (Ligation products) 1ul to DH5α compitent cell.
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| - | #Stood on ice in 30min.
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| - | #Added 600ul of LB to transformed DH5α solution.
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| - | #From 700 solution(100ul is DH5α and 600ul is LB), 100ul add to 900ul of LB(x10 solution)
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| - | #Spread 300ul from 600(700-100)ul and 1000ul of LB&DH5α solution to each LBA plates.
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| - | #Cultivated.
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| - |
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| - | ----
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| - | *9th
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| - |
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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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| - |
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| - | PCR reaction solution
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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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| - | |――――――――――――――――――――――――――
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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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| - | {|
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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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| - | {|
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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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| - |
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| - | ----
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| - | *10th
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| - |
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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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| - |
| |
| - | ;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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| - | {|
| |
| - | |pT7-RBS
| |
| - | |1,5ul
| |
| - | |-
| |
| - | |EcoRI
| |
| - | |1ul
| |
| - | |-
| |
| - | |PstI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xH buffer
| |
| - | |2ul
| |
| - | |-
| |
| - | |DW
| |
| - | |14.5ul
| |
| - | |-
| |
| - | |――――――――――――
| |
| - | |-
| |
| - | |Total
| |
| - | |20ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | Digestion recipe
| |
| - | Ag43-dT
| |
| - | {|
| |
| - | |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]
| |
| - |
| |
| - | ;Digestion
| |
| - | Digestion for 3A Assembly.
| |
| - |
| |
| - |
| |
| - | pT7-RBS
| |
| - | {|
| |
| - | |DNA
| |
| - | |17ul
| |
| - | |-
| |
| - | |EcoRI
| |
| - | |1ul
| |
| - | |-
| |
| - | |SpeI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xH buffer
| |
| - | |3ul
| |
| - | |-
| |
| - | |DW
| |
| - | |8ul
| |
| - | |-
| |
| - | |――――――――――――
| |
| - | |-
| |
| - | |Total
| |
| - | |30ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | Ag43-dT
| |
| - | {|
| |
| - | |DNA
| |
| - | |12.5ul
| |
| - | |-
| |
| - | |XbaI
| |
| - | |1ul
| |
| - | |-
| |
| - | |PstI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xH buffer
| |
| - | |2ul
| |
| - | |-
| |
| - | |DW
| |
| - | |3.5ul
| |
| - | |-
| |
| - | |――――――――――――
| |
| - | |-
| |
| - | |Total
| |
| - | |20ul
| |
| - | |}
| |
| - |
| |
| - |
| |
| - | pSB1C3
| |
| - | {|
| |
| - | |DNA
| |
| - | |20ul
| |
| - | |-
| |
| - | |EcoRI
| |
| - | |1ul
| |
| - | |-
| |
| - | |PstI
| |
| - | |1ul
| |
| - | |-
| |
| - | |10xH buffer
| |
| - | |3ul
| |
| - | |-
| |
| - | |DW
| |
| - | |5ul
| |
| - | |-
| |
| - | |――
| |
| - | |-
| |
| - | |Total
| |
| - | |30ul
| |
| - | |}
| |
| - |
| |
| - | Reacted in 2hrs at 37c.
| |
"
