Template:Kyoto/Notebook/Sep 22

From 2013.igem.org

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(→Transformation)
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!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
|-
|-
-
|experiment||9/13 Pcon-PT181 attenuator (SpeI & PstI)||1.7 µL||9/18 aptamer-1R-DT (XbaI & PstI)||5.4µL|| 7.1µL
+
|experiment||9/13 Pcon-PT181 attenuator (SpeI & PstI)||1.7 µL||9/18 aptamer-1R-DT (XbaI & PstI)||5.4µL|| 3.5µL
|-
|-
-
|experiment||9/12 Plac(SpeI & PstI)||2.4 µL||9/18 aptamer-1R-DT (XbaI & PstI)||6.1µL||8.5 µL
+
|experiment||9/12 Plac(SpeI & PstI)||2.4 µL||9/18 aptamer-1R-DT (XbaI & PstI)||6.1µL||4.5 µL
|-
|-
-
|experiment||9/13 Ptet(SpeI & PstI)||1.7 µL||9/10 Plux-RBS-GFP-DT||7.0 µL||8.7 µL
+
|experiment||9/13 Ptet(SpeI & PstI)||1.7 µL||9/10 Plux-RBS-GFP-DT||7.0 µL||4.4 µL
|-
|-
-
|experiment||9/21 RBS-GFP-DT (EcoRI & XbaI)||0.6 µL||9/18 aptamer 12-1R-DT (XbaI & PstI)||5.6µL||6.2 µL
+
|experiment||9/21 RBS-GFP-DT (EcoRI & XbaI)||0.6 µL||9/18 aptamer 12-1R-DT (XbaI & PstI)||5.6µL||3.1 µL
|-
|-
-
|experiment||9/13 PSB1C3(XbaI & PstI)||1.9 µL||9/16 PT181 attenuator(XbaI & PstI)||4.6µL||6.5 µL
+
|experiment||9/13 PSB1C3(XbaI & PstI)||1.9 µL||9/16 PT181 attenuator(XbaI & PstI)||4.6µL||3.3 µL
|-
|-
|}
|}

Revision as of 09:40, 26 September 2013

Contents

Sep 22

Colony PCR

Tatsui

Samplebase pair
9/20 Plac-PT181 attenuator(1~4)664
9/20 Plac-PT181 antisense(1~6)369
9/20 Pcon-PT181 attenuator-aptamer-DT(1~6)859
9/20 Pcon-PT181 antisense-spinach-DT(1~8)723

Miniprep

Honda

DNAconcentration[µg/mL]260/280260/230
9/21 spinach-DT197.91.671.54
9/21 Plux125.91.711.27
9/21 Plux-RBS-GFP-DT-(1)168.01.621.73
9/21 Pcon-PT181 attenuator-(1)368.31.571.62
9/21 RBS-GFP-DT532.81.751.78
9/21 Pcon-RBS-GFP-DT845.22.021.70

Colony PCR

Honda

Samplebase pair
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1~2)283
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s2min10s30cycles

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
2~4Pcon-aptamer12-1R-DT(E-1A)--
6~8Pcon-spinach-DT(E-1A)--
10~12Pcon-attenuator PT181(E-1A)--
LaneDNAEnzyme
1100bp ladder--
2~4DT(1-3)--
6~8aptamer12-1RDT(1-3)--
10~12Pcon-PT181 attenuator(1-5A)--
Nameconcentration[µg/mL]260/280260/230
Pcon-aptamer12-1R-DT(E-1A)31.21.870.30
Pcon-spinach-DT(E-1A)25.61.880.99
Pcon-attenuator PT181(E-1A)25.31.990.86
DT(1-3)22.11.820.20
aptamer12-1RDT(1-3)33.51.990.05
Pcon-PT181 attenuator(1-5A)36.91.120.96

digestion

No name

9/16 PlacXbaISpeIPstIBufferBBSAMilliQtotal
2cuts12.5µL0µL1µL1µL3µL3µL9.5µL30µL
NC0.8µL0µL0µL0µL1µL1µL7.4µL10µL
9/22 PluxXbaISpeIPstIBufferBBSAMilliQtotal
2cuts16µL0µL1µL1µL3µL3µL14µL30µL
NC0.8µL0µL0µL0µL1µL1µL7.2µL10µL
9/20 RBS-tetR-2R-DT XbaISpeIPstIBufferDBSAMilliQtotal
2cuts8.5µL1µL0µL1µL3µL3µL13.5µL30µL
NC0.4µL0µL0µL0µL1µL1µL7.6µL10µL

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
2~4Pcon-attenuator PT181(E-0)--
LaneDNAEnzyme
1100bp ladder--
2~4Pcon-attenuator PT181(E-0)--
LaneDNAEnzyme
2100bp ladder--
4~6Pcon-attenuator PT181(2-SA)--
LaneDNAEnzyme
1100bp ladder--
3~5spinach-DT(3-2)--
8~10Ptet-antisense PT181(1-3)--
Nameconcentration[µg/mL]260/280260/230
Pcon-attenuator PT181(E-0)2.41.800.17
Pcon-attenuator PT181(2-SA)10.81.850.54
spinach-DT(3-2)36.71.800.75
Ptet-antisense PT181(1-3)16.81.740.78

Colony PCR

No name

Samplebase pair
9/20 Pcon-PT181 attenuator-aptamer12-1R-DT-(7~14)859

Colony PCR

Tatsui

Samplebase pair
PconPT181antisense-spinach-DT(9~15)723


Ligation

No name

stateVectorInserterLigation High ver.2
experiment9/13 Pcon-PT181 attenuator (SpeI & PstI)1.7 µL9/18 aptamer-1R-DT (XbaI & PstI)5.4µL 3.5µL
experiment9/12 Plac(SpeI & PstI)2.4 µL9/18 aptamer-1R-DT (XbaI & PstI)6.1µL4.5 µL
experiment9/13 Ptet(SpeI & PstI)1.7 µL9/10 Plux-RBS-GFP-DT7.0 µL4.4 µL
experiment9/21 RBS-GFP-DT (EcoRI & XbaI)0.6 µL9/18 aptamer 12-1R-DT (XbaI & PstI)5.6µL3.1 µL
experiment9/13 PSB1C3(XbaI & PstI)1.9 µL9/16 PT181 attenuator(XbaI & PstI)4.6µL3.3 µL

Liquid Culture

No name

Samplemedium
9/20 Plac-antisense-1--
9/20 Plac-attenuator-1--
9/20 Plac-antisense-1--
9/20 Plac-attenuator-1--

digestion

No name

9/8 Pcon-PT181 antisenseEcoRISpeI BufferMMilliQtotal
2cuts8.3µL1µL1µL3µL16.7µL30µL
NC0.4µL0µL0µL1µL8.3µL10µL
9/7 Pcon-RBS-tetR-DTEcoRIXbaISpeIPstIBufferDBSAMilliQtotal
2cut14µL1µL1µL0µL0µL3µL18.2µL30µL
NC0.7µL0µL0µL0µL0µL1µL1µL7.3µL10µL
9/22 RBS-GFP-DTEcoRIXbaISpeIPstI BufferDBSAMilliQtotal
2cuts3.8µL1µL1µL0µL0µL3µL3µL18.2µL30µL
NC0.2µL0µL0µL0µL0µL1µL1µL7.8µL10µL
9/6 aptamer12-1R-DTEcoRIXbaISpeIPstIBufferDBSAMilliQ
2cut8µL1µL1µL0µL0µL3µL3µL16µL
NC0.4µL0µL0µL0µL0µL1µL1µL7.6µL
incubate 37°C 1hour

Gel Extraction

No name

LaneDNAEnzyme
11kbp ladder--
3~5Pcon-PT181 antisense (EcoRI&SpaI)--

Transformation

No name

NameSampleCompetent CellsPlate
9/20 Plac-aptamer12-1R-DT(XbaI&PstI)2 µL20 µLAmp
9/20 pT181 attenuator(1C3)(EcoRI&SpeI)2 µL20 µLCP
9/17 pT181 attenuator(1C3)(XbaI&PstI)2 µL20 µLCP
9/20 Pcon-pT181 attenuator(SpaI&PstI)＋aptamer12-1R-DT(XbaI&PstI)2 µL20 µLAmp
kaiABC2 µL20 µLAmp
9/22 pSB1C3-RpaB(XbaI&PstI)2 µL20 µLAmp
9/22 pSB1C3-pT181 attenuator(XbaI&PstI) 2 µL20 µLAmp
9/22 Ptet(SpaI&PstI)＋RBS-GFP-DT(XbaI&PstI)2 µL20 µLCP
9/22 RBS-GFP-DT＋Pcon-attenuator pT181 (EcoRI&SpeI)2 µL20 µLCP
9/22 pT181 attenuator(XbaI&PstI)(1C3)2 µL20 µLCP

PCR

No name

Samplebase pair
Pcon-RBS-tetR-DT(1-2Ⓐ)--
Pcon-RBS-tetR-DT(3-2Ⓐ)--
Pcon-RBS-GFP-DT(1-SⒶ)--
Pcon-RBS-GFP-DT(2-SⒶ)--
Pcon-tetR-DT(E-2Ⓐ)--
PreDenatureDenatureAnnealingExtensioncycle
94 °C94 °C57 °C68 °C--
2 min10 s30 s1min5s30 cycles

Transformation

Tatsui

NameSampleCompetent CellsPlate
9/14 pSB1C3-Pcon-pT181 attenuator-DT2 µL20 µLCP
9/14 pSB1C3-Pcon-aptamer 12_1R-DT2 µL20 µLCP
9/14 pSB1C3-Pcon-antisense-spinach-DT2 µL20 µLCP
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