Template:Kyoto/Notebook/Sep 4

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==Sep 4==
==Sep 4==
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===Ligation===
===Ligation===
<div class="experiment">
<div class="experiment">

Revision as of 12:31, 25 September 2013

Contents

Sep 4

Ligation

No name

stateVectorInserterLigation High ver.2
experiment9/3 DT(EcoRI&XbaI)3.2µL9/3 RBS-lysis12.4µL2.7µL
experiment9/3 DT(EcoRI&XbaI) 3.0µL 9/3 RBS-lysis24.7µL 4.0µL
experiment9/3 DT(EcoRI&XbaI) 3.0µL 9/3 RBS-lysis38.8µL 4.0µL
experiment9/3 DT(EcoRI&XbaI) 3.0µL 9/3 aptamer12_1R(EcoRI&SpeI) 1.9µL 2.45µL
experiment9/2 pSB1C3(XbaI&PstI)10.6µL 9/3 pT181attenuator (XbaI& PstI)3.2µL 4.0µL
experiment9/2 pSB1C3(XbaI&PstI)9.6µL 9/3 pT181antisense (XbaI& PstI)2.1µL 4.0µL
experiment9/2 pSB1C3(EcoRI&SpeI)16.7µL 9/3 aptamer12_1R(EcoRI&SpeI) 2.0µL 4.0µL

incubate 16 °C 1 hour

Transformation

Tatsui

NameSampleCompetent CellsPlate
9/4 RBS-lysis1-DT 2µL20µLAmp
9/4 RBS-lysis2-DT 2µL20µLAmp
9/4 RBS-lysis3-DT 2µL20µLAmp
9/4 aptamer12_1R-DT2µL20µLAmp
9/4 pT181attenuator –pSB1C32µL20µLAmp
9/4 pT181antisense-pSB1C3 2µL20µLAmp
9/4 aptamer12_1R –pSB1C32µL20µLAmp
9/4 pSB4K5(2013plate5 56) 2µL20µLAmp
9/3 Mutagenesis product 1(KaiABC) 2µL20µLAmp
9/3 Mutagenesis product 2(NC) 2µL20µLAmp

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
DT193.11.931.81
Spinach-DT373.31.921.69
RBS-lysis1332.01.951.62

Master Plate

Hirano

NumberUse LB plate (+CP)
18/10 Plac(CP)-2
2 8/10 Plac(CP)-2
38/10 Plac(CP)-2
NumberUse LB plate (+Amp)
18/9 J23100(Amp)-1
28/9 J23100(Amp)-1
38/9 J23100(Amp)-1

Liquid Culture

Hirano

Samplemedium
8/10 Plac(CP)-2Plusgrow medium(+CP)
8/9 J23100(Amp)-1Plusgrow medium(+Amp)

PCR

Hirano

8/29 Pλ-RBS-luxI-DTKOD plus10x bufferdNTPMgSO4F primer (RBS-luxI-DT-cloning-fwd) R primer(RBS-luxI-DT-cloning-rev)MilliQtotal
0.3µL0.5µL 2.5µL 2.5µL 1.5µL 0.75µL 0.75µL 16.2µL 25µL
8/31 Pbad/araC-RBS-RFP(1)KOD plus10x bufferdNTPMgSO4F primer (Pbad/araC-pSB1C3-cloning-fond) R primer(Pbad/araC-cloning-rex)MilliQtotal
0.3µL0.5µL 2.5µL 2.5µL 1.5µL 0.75µL 0.75µL 16.2µL 25µL
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C65°C68°C--
5min10s30s3min10s30cycles

Restriction Enzyme Digestion

No name

8/21 PconstSpeIPstIBufferBSAMilliQtotal
2 cuts10.5µL1.0µL1.0µL3.0µL0.3µL14.2µL30 µL
NC0.5µL0µL0µ1.0µL0.1µL8.4µL10 µL
8/29 PlacSpeIPstIBufferBSAMilliQtotal
2 cuts13.0µL1.0µL1.0µL3.0µL0.3µL11.7µL30 µL
NC0.7µL0µL0µ1.0µL0.1µL 8.2µL10 µL
8/20 PtetSpeIPstIBufferBSAMilliQtotal
2 cuts9.0µL1.0µL1.0µL3.0µL0.3µL15.7µL30 µL
NC0.5µL0µL0µ1.0µL0.1µL 8.4µL10 µL
8/20 RBS-tetR-DTXbaIPstIBufferBSAMilliQtotal
2 cuts8.5µL1.0µL1.0µL3.0µL0.3µL16.2µL30 µL
NC0.5µL0µL0µ1.0µL0.1µL 8.4µL10 µL
spinach -DTXbaIPstIBufferBSAMilliQtotal
2 cuts5.4µL1.0µL1.0µL3.0µL3.0µL16.6µL30 µL
NC0.3µL0µL0µ1.0µL1.0µL 7.7µL10 µL
8/17 RBS-lacZα-DT XbaIPstIBufferBSAMilliQtotal
2 cuts11µL1.0µL1.0µL3.0µL3.0µL11µL30 µL
NC0.5µL0µL0µ1.0µL1.0µL7.5µL10 µL

Transformation

No name

NameSampleCompetent CellsTotalPlate
Ptrc KaiC1µL10µL11µLAmp
Ptrc KaiB1µL10µL11µLAmp
pSΩ 1µL10µL11µLAmp

Electrophoresis

No name
1

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2PconstSpeIPstI
3Pconst NC----
4PlacSpeIPstI
5Plac NC----
6100bp ladder----

2

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2PtetSpeIPstI
3Ptet NC----
4RBS-tetR-DTXbaIPstI
5RBS-tetR-DT NC----
6100bp ladder----

Igku Sep4 Electrophoresis(N1).jpg

Gel Extraction

No name

1

LaneDNAEnzyme
1100bp ladder--
2PconSpeI&PstI
3PconSpeI&PstI
4----
5PlacSpeI&PstI
6PlacSpeI&PstI

2

LaneDNAEnzyme
1100bp ladder--
2PtetSpeI&PstI
3PtetSpeI&PstI
4----
5RBS-tetR-DTXbaI&PstI
6RBS-tetR-DTXbaI&PstI

Igku Sep4 Electrophoresis(N2).jpg
Igku Sep4 Gel Extraction(N2) 2.jpg

Namequantityconcentration[µg/mL]260/280260/230
Pcon--13.2 1.99 -1.64
Plac-- 15.3 2.07 -0.39
Ptet-- 30.11.97 -0.97
RBS-tetR-DT--3.02.44 0.88

Gel Extraction

No name
LaneDNAEnzyme
1100bp ladder--
2spinach-DTXbaI&PstI
3spinach-DT XbaI&PstI
4----
5RBS-lacZα-DTXbaI&PstI
6RBS-lacZα-DTXbaI&PstI

Igku Sep4 Gel Extraction(N3) 1.jpg
Igku Sep4 Gel Extraction(N3) 2.jpg

Namequantityconcentration[µg/mL]260/280260/230
spinach-DT--2.12.10-0.05
RBS-lacZα-DT--3.32.59-0.07

Master Plate

No name

NumberUse LB plate (+Amp)
48/9 BBa_J23100-3
58/9 BBa_J23100-4
68/9 BBa_J23100-5
NumberUse LB plate
1JM109-1
2JM109-2
3JM109-3
4JM109-4

Liquid Culture

Hirano

Samplemedium
8/9 BBa_J23100-3Plusgrow medium(+Amp)
JM109-1(Master plate)LB medium

Colony PCR

Hirano

Samplebase pair
8/9 BBa_J23100 3--
8/9 BBa_J23100 4--
8/9 BBa_J23100 5--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30s30cycles
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