Template:Kyoto/Notebook/Sep 11

From 2013.igem.org

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===Miniprep===
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===Liquid Culture===
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<span class="author">Nakamoto</span>
<span class="author">Nakamoto</span>
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{| class="wikitable"
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!DNA||concentration[&micro;g/mL]||260/280||260/230
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!Sample||medium
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|Pcon-pT181attenuater-DT||293.6　||1.88　||1.84
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|9/10Pcon-aptamer12_1R-DT1||amp
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|Pcon-apt12_1R-DT||303.8　||1.75　||1.64
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|DT+Pcon-pT181 attenuater1||cp
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|Fusion1attenuater(p3B1C3)||172.0　||1.96　||2.31
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|Fusion1 attenuater(pSB1C3)-1||cp 　
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|RBS-lysis1-DT||235.1　||1.59　||1.60
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|RBS-lysis2-DT||387.7　||1.91　||2.01
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|RBS-lysis3-DT||387.9　||1.91　||2.09
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|Plux||166.7　||1.98　||1.63
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Revision as of 14:24, 24 September 2013

Contents

Sep 11

PCR

Tatsui

pKaiBC(200p)10*buffer KOD plusdNTPsMgSO4primer mix(F&R)KOD plusMilliQtotal
0.52.52.51.51.50.51625
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　60°C　68°C30
2min　10sec　30sec　30sec　
pKaiBC(400p)10*buffer KOD plusdNTPsMgSO4primer mix(F&R)KOD plusMilliQtotal
12.52.51.51.50.515.525
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　60°C　68°C30
2min　10sec　30sec　30sec　
pKaiBC(800p)10*buffer KOD plusdNTPsMgSO4primer mix(F&R)KOD plusMilliQtotal
22.52.51.51.50.514.525
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　60°C　68°C30
2min　10sec　30sec　30sec　
pKaiBC(1ng)10*buffer KOD plusdNTPsMgSO4primer mix(F&R)KOD plusMilliQtotal
2.52.52.51.51.50.51425
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　60°C　68°C30
2min　10sec　30sec　30sec　

colony PCR

Tatsui

Samplebase pair
9/9Pcon-lacZα(p3B4k5)712bp
9/9Pcon-spinach-DT(p3B4k5)605bp
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
5min30s　30s　42s　
Samplebase pair
Pbad\araC-RBS-luxI-DT
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
5min30s　30s　2min　
Samplebase pair
9/9Pcon+RBS-luxI-DT1079bp
9/10Pcon+sptamer12_1R-DT584bp
9/10DT+Pcon-pT181 attenuater 1781bp
9/10Fusion1 attenuater(p3B1C3)-1638bp
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
50sec　30sec　30sec　1min　

Restriction Enzyme Digestion

Nakamoto

DNAEcoRlXbalSpelPstlbufferBSAMilliQTotal
pSB4K5(EcoRl+Spel)9.4µL 1µL 0µL 1µL 0µL 3µL 3µL 12.6µL30 µL
pSB4K5(EcoRl+Spel) negative0.5µL 0µL 0µL 0µL 0µL 1µL 1µL 7.5µL 10µL
RBS-lacZα-DT(EcoRl+Spel) 10µL 1µL 0µL 1µL 0µL 3µL 3µL 12µL 30µL
RBS-lacZα-DT(EcoRl+Spel) negative 0.5µL 0µL 0µL 0µL 0µL 1µL 1µL 7.5µL 10µL
Ptet-pT181 antisense(Stel+Pstl) 10.8µL 0µL 0µL 1µL 1µL 3µL 3µL 12µL 30µL
Ptet-pT181 antisense(Stel+Pstl) negative 0.5µL 0µL 0µL 0µL 0µL 1µL 1µL 7.5µL 10µL
Pcon-RBS-lacZα-DT(EcoRl+Spel) 7.1µL 1µL 0µL 1µL 0µL 3µL 3µL 14.9µL 30µL
Pcon-RBS-lacZα-DT(EcoRl+Spel) negative 0.4µL 0µL 0µL 0µL 0µL 1µL 1µL 7.6µL 10µL
RBS-lysis2-DT(Xbal+Pstl) 22µL 0µL 1µL 0µL 1µL 3µL 3µL 0µL 30µL
RBS-lysis2-DT(Xbal+Pstl) negative 1.4µL 0µL 0µL 0µL 0µL 1µL 1µL 6.6µL 10µL

Liquid Culture

Nakamoto

Samplemedium
8/16 Master4 Plax
8/25 Pλ-laxI-2 　

PCR

Hirano

8/29 Pλ-RBS-luxI-DT(76.5ng/µL)10x buffer for KOD plus-ver.2KOD-plusdNTPMgSO4F primer(RBS-luxI-DT-cloning-fwd)R primer(RBS-luxI-DT-cloning-rev)MilliQtotal
0.32.50.52.51.50.750.7516.225
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　65°C　68°C--
5min30sec30sec1min30
8/31 Pbad/araC-RBS-RFP(1)(67.6ng/µL)10x buffer for KOD plus-ver.2KOD-plusdNTPMgSO4F primer(Pbad/araC(pSBlc3)-cloning-fwd)R primer(Pbad/araC(pSBlc3)-cloning-rev)MilliQtotal
0.32.50.52.51.50.750.7516.225
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　54°C　68°C--
5min30sec30sec3min 20sec30

Liquid Culture

Hirano

Samplemedium
9/4 RBS-lysis1+DT 3plusgrow(+CP)
9/4 RBS-lysis2+DT 1plusgrow(+CP)
9/4 RBS-lysis3+DT 3plusgrow(+CP)

Liquid Culture

Nakamoto

Samplemedium
9/10Pcon-aptamer12_1R-DT1amp
DT+Pcon-pT181 attenuater1cp
Fusion1 attenuater(pSB1C3)-1cp 　
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