Template:Kyoto/Notebook/Sep 12

From 2013.igem.org

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|2||RBS-lacZ-DT||EcoR1&Spe1
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|2||RBS-lacZα-DT||EcoRI&SpeI
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|3||RBS-lacZ-DT||EcoR1&Spe1
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|3||RBS-lacZα-DT||EcoRI&SpeI
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|4||RBS-lacZ-DT||EcoR1&Spe1
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|4||RBS-lacZα-DT||EcoRI&SpeI
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|5||--||--
|5||--||--
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|6||Pcon-RBS-lacZ-DT||EcoR1&Spe1
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|6||Pcon-RBS-lacZα-DT||EcoRI&SpeI
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|7||Pcon-RBS-lacZ-DT||EcoR1&Spe1
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|7||Pcon-RBS-lacZα-DT||EcoRI&SpeI
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|8||Pcon-RBS-lacZ-DT||EcoR1&Spe1
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|8||Pcon-RBS-lacZα-DT||EcoRI&SpeI
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!Name||concentration[µg/mL]||260/280||260/230
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|RBS-lacZ-DT(EcoR1&Spe1)||9.8||1.50||0.39
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|RBS-lacZα-DT(EcoRI&SpeI)||9.8||1.50||0.39
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|Pcon-RBS-lacZ-DT(EcoR1&Spe1)||11.2||1.47||0.08
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|Pcon-RBS-lacZα-DT(EcoRI&SpeI)||11.2||1.47||0.08
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Revision as of 08:45, 24 September 2013

Contents

Sep 12

Miniprep

Nakamoto

DNAconcentration[µg/mL]260/280260/230
Pcon-pT181atte-DT293.6　1.88　1.84
Pcon-apt12-1R-DT303.8　1.75　1.64
Fusion1atte(pSB1c3)172.0　1.96　2.31
RBS-lisis1-DT235.11.591.60
RBS-lisis2-DT387.71.912.01
RBS-lisis3-DT387.91.912.09
Plux166.71.981.63

Electrophoresis

Nakamoto

LaneSample
1100bp ladder
29/11 P-RBS-lisis1-DT(Colony PCR prodution)

File:Igku xxxxxx.xxx

Liquid Culture

Nakamoto

Samplemedium
9/10 Pcon-apt12-1R-DT1Plusgrow medium(+Amp)
9/10 DT-Pcon-pT181atte1Plusgrow medium(+CP)
9/10 Fusion1-atte(pSB1c3)Plusgrow medium(+CP)　

Electrophoresis

Tatsui

1

LaneSample
11kb ladder
2pSB4K5(EcoRI&SpeI)
3 pSB4K5 NC(EcoRI&SpeI)
4RBS-lacz-DT(EcoRI&SpeI)
5 EcoR1&Spe1 NC( EcoRI&SpeI)
6Pcon-RBS-lacz-DT( EcoRI&SpeI)
7 Pcon-RBS-lacz-DT NC( EcoRI&SpeI)
81kb ladder

File:Igku xxxxxx.xxx

2

LaneSample
11kb ladder
2Ptet-pT181anti(SpeI&PstI)
3 Ptet-pT181anti NC(SpeI&PstI)
4RBS-lysis2-DT(XbaI&PstI)
5 EcoR1&Spe1 NC( EcoRI&SpeI)
6--
7--
81kb ladder

File:Igku xxxxxx.xxx

Gel Extraction

Tatsui

LaneDNAEnzyme
11kb ladder--
2RBS-lacZα-DTEcoRI&SpeI
3RBS-lacZα-DTEcoRI&SpeI
4RBS-lacZα-DTEcoRI&SpeI
5----
6Pcon-RBS-lacZα-DTEcoRI&SpeI
7Pcon-RBS-lacZα-DTEcoRI&SpeI
8Pcon-RBS-lacZα-DTEcoRI&SpeI
91kb ladder--

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(EcoRI&SpeI)9.81.500.39
Pcon-RBS-lacZα-DT(EcoRI&SpeI)11.21.470.08

Gel Extraction

Tatsui

LaneDNAEnzyme
11kb ladder--
2RBS-lysis2-DTXba1&Pst1
3RBS-lysis2-DTXba1&Pst1
4RBS-lysis2-DTXba1&Pst1
5----
61kb ladder--

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lysis2-DT(Xba1&Pst1)5.41.430.77

Electrophoresis

No name


LaneSample
11kb ladder
2Pλ-RBS-luxI-DT(PCR product)
3Pbad/araC-RBS-RFP(PCR product)


Colony PCR

Honda

Samplebase pair
9/11 apt12-1M(pSB1C3) 1513
9/11 apt12-1M(pSB1C3) 2513
9/11 Fusion3m2a09ttenuator(pSB1C3) 1609
9/11 Fusion6 antisense(pSB1C3)1431-
9/11 aptamer 12-P(pSB1C3)1515
9/11 Fusion1 antisense(pSB1C3) 1420
9/11 Fusion1 antisense(pSB1C3) 2420
9/11 Plac(BBa-R0011) 1293
9/11 Plac(BBa-R0011) 2293
9/11 Plac(BBa-R0011) 3293
9/11 Plac(BBa-R0011) 4293
9/11 Pcon-luxR-Plux-GFP 12138
9/11 Pcon-RBS-lacZα-DT(pSB4K5) 1712
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s2min12s30cycles

Restriction Enzyme Digestion

Tatsui

9/5 Pcon EcoRISpeIXbaIPstI BufferBSAMilliQtotal
1cuts(P)5.70µL0µL0µL1µL3µL3µL17.3µL30µL
NC(P)1.90µL0µL0µL0µL1µL1µL6.1µL10µL
8/17 DTEcoR1BufferBSAMilliQtotal
1cut10.61µL3µL3µL12.4µL30µL
NC3.50µL1µL1µL4.5µL10µL

Colony PCR

Nakamoto,Tatsui

Samplebase pair
9/11 Pcon-pT181atte-RBS-lacZα-DT965
9/11 Plux-PBS-lysis1-DT 1613
9/11 Pcon-pT181attenuator-aptamer12-1R-DT 1859
9/11 Pcon-pT181attenuator-aptamer12-1R-DT 2859
9/11 Plux-RBS-lysis3-DT 11210
9/11 Plux-RBS-lysis3-DT 21210
Pcon-Spinach-DT(pSB4K5) 1605
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min12s30cycles

Transformation

tatsui

NameSampleCompetent CellsPlate
9/7 pSB4K52µL20µLAmp

Electrophoresis

No name

1

LaneSample
1Fusion6 antisense
2Fusion1 antisense-1
3 Fusion1 antisense-2
4100bp ladder
5Plac(BBa-R0011) 1
6 Plac(BBa-R0011) 2
7 Plac(BBa-R0011) 3
8 Plac(BBa-R0011) 4

File:Igku xxxxxx.xxx

2

LaneSample
11kb ladder
2aptamer12-1M 1
3 aptamer12-1M 2
4Fusion3m2 attenuator
5aptamer12-P1
6Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT
7Pcon-RBS-lacZα=DT
8 1kb ladder

Electrophoresis

No name

LaneSampleEnzyme
1Pcon-pT181attenuator-RBS-lacZα-DT 1--
2Pcon-pT181attenuator-RBS-lacZα-DT 2--
3Plux-RBS-lysis1-DT 1 --
4Pcon-pT181attenuator-aptamer12-1R-DT 1 --
5Pcon-pT181attenuator-aptamer12-1R-DT 2 --
6Plux-RBS-lysis3-DT 1--
71kbp ladder --
8 Plux-RBS-lysis3-DT 2--
9Pcon-spinach-DT(pSB4K5) 1--
109/5 PconPst1
119/5 Pcon NC--
128/17 DT EcoR1
138/17 DT --

Liquid Culture

Nakamoto

Samplemedium
9/11Fusion6 antisense-1Plusgrow medium(+CP)
Plac(BBa-R0011)-3Plusgrow medium(+Amp)
Plac(BBa-R0011)-4Plusgrow medium(+Amp)
Fusion3m2 attenuator -1Plusgrow medium(+CP)　
Pλ-luxI-2Plusgrow medium(+Amp)　
9/11 aptamer12-1M-2Plusgrow medium(+CP)

Colony PCR

Nakamoto

Samplebase pair
9/11 Fusion1 antisense 3394
9/11 Fusion1 antisense 4394
9/11 aptamer12-P 2378
9/11 aptamer12-P 3378
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s36s30cycles

File:Igku Aug19electrophoresis1.png

Electrophoresis

Nakamoto

LaneSampleEnzyme
1Fusion1 anti 2 --
2 Fusion1 anti 3--
31kb ladder --
4apt12-P 2 --
5apt12-P 3 --

Restriction Enzyme Digestion

tatsui

9/12 RBS-lysis2-DTXba1 PstIBufferBSAMilliQtotal
2 cuts5.2 1.0µL1.0µL3.0µL3.0µL16.8µL 30µL
NC0.3µL0µL0µL1.0µL1.0µL7.7µ10µL
9/12 RBS-lysis3-DTXba1 PstIBufferBSAMilliQtotal
2 cuts5.2 1.0µL1.0µL3.0µL3.0µL16.8µL 30µL
NC0.3µL0µL0µL1.0µL1.0µL7.7µ10µL
9/11 RBS-lysis1-DTXba1 PstIBufferBSAMilliQtotal
2 cuts8.51.0µL1.0µL3.0µL3.0µL13.5µL 30µL
NC0.4µL0µL0µL1.0µL1.0µL7.6µ10µL
8/20 J23100-RBS-luxR -DTEcoR1 BufferBSAMilliQtotal
1cut5.81.0µL3.0µL3.0µL17.2µL 30µL
NC0.3µL0µL1.0µL1.0µL7.7µ10µL
9/10 pSB1C3EcoR1 Spe1BufferBSAMilliQtotal
2cuts8.11.0µL1.0µL3.0µL3.0µL13.9µL 30µL
NC0.4µL0µL0µL1.0µL1.0µL7.6µ10µL
9/12 Plux Pst1 BufferBSAMilliQtotal
1cut12.01.0µL3.0µL3.0µL11.0micro;L 30µL
NC0.6µL0µL1.0µL1.0µL7.4µ10µL
9/10 pSB1C3Xba1 Pst1BufferBSAMilliQtotal
2cuts8.11.0µL1.0µL3.0µL3.0µL13.9µL 30µL

Electrophoresis

Tatsui

LaneSampleEnzyme1Enzymel2
19/12 RBS-lysis2-DT Xba1Pst1
29/12 RBS-lysis2-DT NC ----
39/11 RBS-lysis1-DT Xba1Pst1
49/11 RBS-lysis1-DT NC ----
5 9/11 RBS-lysis3-DT Xba1Pst1
6 9/11 RBS-lysis3-DT NC1----
71kb ladder ----
8 100bp ladder----
9J23100-RBS-luxR-DT EcoR1--
10 J23100-RBS-luxR-DT ----
119/10 pSB1C3 EcoR1Spe1
12 9/10 pSB1C3 NC-----
139/10 pSB1C3 Xba1Pst1
149/12 PluxPst1--
159/12 Plux NC----
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