Template:Kyoto/Notebook/Sep 12

From 2013.igem.org

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Revision as of 16:30, 23 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)　
v>

Electrophoresis

Tatsui

1

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

File:Igku xxxxxx.xxx

2

LaneSample
11kb ladder
2Ptet-pT181anti(Spe1&Pst1)
3 Ptet-pT181anti NC(Spe1&Pst1)
4RBS-lysis2-DT(Xba1&Pst1)
5 EcoR1&Spe1 NC( EcoR1&Spe1)
6--
7--
81kb ladder

File:Igku xxxxxx.xxx

Gel Extraction

Tatsui

LaneDNAEnzyme
11kb ladder--
2RBS-lacZ-DTEcoR1&Spe1
3RBS-lacZ-DTEcoR1&Spe1
4RBS-lacZ-DTEcoR1&Spe1
5----
6Pcon-RBS-lacZ-DTEcoR1&Spe1
7Pcon-RBS-lacZ-DTEcoR1&Spe1
8Pcon-RBS-lacZ-DTEcoR1&Spe1
91kb ladder--

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZ-DT(EcoR1&Spe1)9.81.500.39
Pcon-RBS-lacZ-DT(EcoR1&Spe1)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

Colony PCR

Nakamoto,Tatsui

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

File:Igku xxxxxx.xxx

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

</div>

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
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