Team:HokkaidoU Japan/Notebook/August27

From 2010.igem.org

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<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/August26|August 26]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/August30|August 30]]</div></div>
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=Transfer [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-1A]] on pSB1A10 to pSB1C3=
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1.estimated concentrations of pSB1C3 and [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-1A]] which was amplified by miniprep before
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2.mixed solutions according to the table below
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<div style="float:left;">
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{|style="text-align: center;" class="protocol"
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|-
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!Reagent
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!Amount
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|-
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|pSB1C3
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|5 uL
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|-
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|DW
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|75 uL
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|-
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|10x M Buffer
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|10 uL
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|-
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|0.1%BSA
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|10 uL
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|-
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|EcoRI
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|5 uL
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|-
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|PstI
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|5 uL
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|-
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|style="border-top:1px solid #996"|'''Total'''
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|style="border-top:1px solid #996"|'''100 uL'''
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|}
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</div>
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<div style="float:left; margin-left:50px;">
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{|style="text-align: center;" class="protocol"
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|-
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!Reagent
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!Amount
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|-
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|1-1A
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|8 uL
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|-
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|DW
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|6 uL
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|-
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|10x M Buffer
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|2 uL
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|-
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|0.1%BSA
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|2 uL
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|-
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|EcoRI
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|1 uL
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|-
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|PstI
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|1 uL
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|-
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|style="border-top:1px solid #996"|'''Total'''
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|style="border-top:1px solid #996"|'''20 uL'''
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|}
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</div>
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3.incubated at 37C for 60min
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4.purified the two samples
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5.added 250 uL 100% EtOH to each samples
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6.centrifuged at 4C, 15000rpm for 10 min
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7.discarded supernatant
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8.put the samples into desiccator
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9.added 10 uL DW to pSB1C3 and 2 uL DW to 1-1A
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10.mixed 1 uL pSB1C3 and 2 uL 1-1A
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11.added 3 uL ligation solution and 0.5 uL T4 ligase
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12.transfered the sample to a tube of 500 uL
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13.incubated at 16C for 30 min
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->For not putting 3M CH3COONa into the solutions,we stopped operation up to this.
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=Preparation of 3 Piece Ligation=
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*Parts Information
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{|border="1" style="margin-left: 20px;" class="protocol"
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|-
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!Name
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!Biobrick Name
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!Well
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!Length
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|-
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| Heat Sensor
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| BBa_K098995
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| 3-1E
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| 937 bp
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|-
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| RBS
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| BBa_B0034
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| 1-2M
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| 12 bp
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|-
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| RFP
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| BBa_E1010
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| 1-18F
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| 681 bp
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|-
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| Double Terminator
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| BBa_0015
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| 1-23L
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| 129 bp
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|}
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1.estimated concentrations of Heat Sensor,RBS,RFP and Double Terminator
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2.mixed solutions according to the table below
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<div style="float:left;">
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{|style="text-align: center; margin-left:20px" class="protocol"
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|-
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!Reagent
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!Amount
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|-
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| DW
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| 13.5 uL
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|-
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| 10x M buffer
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| 5 uL
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|-
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| 0.1%BSA
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| 5 uL
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|-
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| EcoRI
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| 3 uL
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|-
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| SpeI
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| 1 uL
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|-
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| Heat Sensor
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| 22.5 uL
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|-
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|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #996;"|'''50 uL'''
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|}
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</div>
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<div style="float:left;">
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{|style="text-align: center; margin-left:20px" class="protocol"
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|-
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!Reagent
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!Amount
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|-
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| DW
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| 34.9 uL
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|-
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| 10x M buffer
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| 5 uL
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|-
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| 0.1%BSA
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| 5 uL
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|-
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| XbaI
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| 1 uL
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|-
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| PstI
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| 2 uL
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|-
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| RBS
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| 34.9 uL
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|-
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|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #996;"|'''50 uL'''
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|}
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</div>
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<div style="float:left;">
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{|style="text-align: center; margin-left:20px" class="protocol"
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|-
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!Reagent
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!Amount
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|-
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| DW
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| 25.5 uL
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|-
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| 10x M buffer
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| 5 uL
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|-
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| 0.1%BSA
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| 5 uL
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|-
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| EcoRI
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| 3 uL
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|-
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| SpeI
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| 1 uL
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|-
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| RFP
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| 10.5 uL
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|-
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|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #996;"|'''50 uL'''
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|}
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</div>
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<div style="float:left;">
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{|style="text-align: center; margin-left:20px" class="protocol"
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|-
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!Reagent
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!Amount
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|-
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| DW
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| 34 uL
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|-
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| 10x M buffer
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| 5 uL
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|-
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| 0.1%BSA
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| 5 uL
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|-
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| XbaI
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| 1 uL
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|-
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| PstI
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| 2 uL
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|-
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| Double Terminator
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| 3 uL
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|-
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|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #996;"|'''50 uL'''
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|}
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</div>

Latest revision as of 01:13, 28 October 2010

Notebook

Transfer 1-1A on pSB1A10 to pSB1C3

1.estimated concentrations of pSB1C3 and 1-1A which was amplified by miniprep before

2.mixed solutions according to the table below


Reagent Amount
pSB1C3 5 uL
DW 75 uL
10x M Buffer 10 uL
0.1%BSA 10 uL
EcoRI 5 uL
PstI 5 uL
Total 100 uL
Reagent Amount
1-1A 8 uL
DW 6 uL
10x M Buffer 2 uL
0.1%BSA 2 uL
EcoRI 1 uL
PstI 1 uL
Total 20 uL













3.incubated at 37C for 60min

4.purified the two samples

5.added 250 uL 100% EtOH to each samples

6.centrifuged at 4C, 15000rpm for 10 min

7.discarded supernatant

8.put the samples into desiccator

9.added 10 uL DW to pSB1C3 and 2 uL DW to 1-1A

10.mixed 1 uL pSB1C3 and 2 uL 1-1A

11.added 3 uL ligation solution and 0.5 uL T4 ligase

12.transfered the sample to a tube of 500 uL

13.incubated at 16C for 30 min

->For not putting 3M CH3COONa into the solutions,we stopped operation up to this.


Preparation of 3 Piece Ligation

  • Parts Information
Name Biobrick Name Well Length
Heat Sensor BBa_K098995 3-1E 937 bp
RBS BBa_B0034 1-2M 12 bp
RFP BBa_E1010 1-18F 681 bp
Double Terminator BBa_0015 1-23L 129 bp


1.estimated concentrations of Heat Sensor,RBS,RFP and Double Terminator

2.mixed solutions according to the table below

Reagent Amount
DW 13.5 uL
10x M buffer 5 uL
0.1%BSA 5 uL
EcoRI 3 uL
SpeI 1 uL
Heat Sensor 22.5 uL
Total 50 uL
Reagent Amount
DW 34.9 uL
10x M buffer 5 uL
0.1%BSA 5 uL
XbaI 1 uL
PstI 2 uL
RBS 34.9 uL
Total 50 uL













Reagent Amount
DW 25.5 uL
10x M buffer 5 uL
0.1%BSA 5 uL
EcoRI 3 uL
SpeI 1 uL
RFP 10.5 uL
Total 50 uL
Reagent Amount
DW 34 uL
10x M buffer 5 uL
0.1%BSA 5 uL
XbaI 1 uL
PstI 2 uL
Double Terminator 3 uL
Total 50 uL
Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/August27"