Template:Kyoto/Notebook/Sep 3

From 2013.igem.org

Contents

Sep 3

Electrophoresis

Kojima

LaneSample
1100bp ladder
2pT181 attenuator(2)-DT2(738)
3Ptet-RBS-lacZα-DT-2(756)
4Ptet-RBS-lacZα-DT-3
5Ptet-RBS-lacZα-DT-4
6Ptet-RBS-lacZα-DT-5
7Ptet-RBS-lacZα-DT-6
8RBS-lysis2-DT-2(985)
9RBS-lysis2-DT-3
10100bp ladder

Igku Sep3 Electrophoresis(N1).jpg

Miniprep

Tatsui

DNAconcentration[µg/mL]260/280260/230
9/2 14:00 pT181 attenuator418.41.821.80
9/2 14:00 pT181 antisense362.21.921.96
9/2 14:00 Spinach438.31.861.99
9/2 14:00 tetR-apt 12_1R567.51.901.99
9/2 21:00 pT181 attenuator320.91.902.15
9/2 21:00 Spinach457.01.882.24

Liquid Culture

Kojima

Samplemedium
Spinach-DT(9/2 master plate)plusgrow(CP)4ml

Restriction Enzyme Digestion

Kojima

DNAEcoRISpeIXbaIPstI10xBuffer B(EcoRI&SpeI)10xBuffer D(XbaI&PstI)100xBSAMilliQtotal
9/3 pT181attenuator 14:00 418.4ng/µL 4.8µL 1.0µL 1.0µL 0µL 0µL 3µL 0µL 0.3µL 19.9µL 30µL
9/3 pT181attenuator 14:00 418.4ng/µL 0.2µL 0µL 0µL 0µL 0µL 1µL 0µL 0.1µL 8.7µL 10µL
9/3 pT181attenuator 14:00 418.4ng/µL 4.8µL 0µL 0µL 1.0µL 1.0µL 0µL 3µL 0.3µL 19.9µL 30µL
9/3 pT181attenuator 14:00 418.4ng/µL 0.2µL 0µL 0µL 0µL 0µL 0µL 1µL 0.1µL 8.7µL 10µL
DNAEcoRISpeIXbaIPstI10xBuffer B(EcoRI&SpeI)10xBuffer D(XbaI&PstI)100xBSAMilliQtotal
9/3 pT181antisense 14:00 362.2ng/µL 5.5µL 1.0µL 1.0µL 0µL 0µL 3µL 0µL 0.3µL 19.2µL 30µL
9/3 pT181attenuator 14:00 418.4ng/µL 0.3µL 0µL 0µL 0µL 0µL 1µL 0µL 0.1µL 8.6µL 10µL
9/3 pT181attenuator 14:00 418.4ng/µL 5.5µL 0µL 0µL 1.0µL 1.0µL 0µL 3µL 0.3µL 19.2µL 30µL
9/3 pT181attenuator 14:00 418.4ng/µL 0.3µL 0µL 0µL 0µL 0µL 0µL 1µL 0.1µL 8.6µL 10µL
DNAEcoRISpeI10xBuffer B100xBSAMilliQtotal
9/3 apt 12_1R 14:00 507.5ng/µL 3.9µL 1.0µL 1.0µL 3µL 0.3µL 20.8µL 30µL
9/3 apt 12_1R 14:00 507.5ng/µL 0.2µL 0µL 0µL 1µL 0.1µL 8.7µL 10µL
DNAEcoRIXbaI10xBuffer D100xBSAMilliQtotal
8/29 DT 166.2ng/µL 12µL 1.0µL 1.0µL 3µL 0.3µL 12.7µL 30µL
8/29 DT 166.2ng/µL 0.6µL 0µL 0µL 1µL 0.1µL 8.3µL 10µL
DNAEcoRISpeI10xBuffer B100xBSAMilliQtotal
8/22 RBS-lysis1(2) 96ng/µL 21µL 1.0µL 1.0µL 3µL 0.3µL 3.7µL 30µL
8/22 RBS-lysis1(2) 96ng/µL 1.0µL 0µL 0µL 1µL 0.1µL 7.9µL 10µL
DNAEcoRISpeI10xBuffer B100xBSAMilliQtotal
8/30 RBS-lysis2 220ng/µL 9.1µL 1.0µL 1.0µL 3µL 0.3µL 15.6µL 30µL
8/30 RBS-lysis2 220ng/µL 0.5µL 0µL 0µL 1µL 0.1µL 8.4µL 10µL
DNAEcoRISpeI10xBuffer B100xBSAMilliQtotal
8/20 RBS-lysis3(1) 282ng/µL 7.1µL 1.0µL 1.0µL 3µL 0.3µL 17.6µL 30µL
8/20 RBS-lysis3(1) 282ng/µL 0.4µL 0µL 0µL 1µL 0.1µL 8.5µL 10µL

PCR

Hirano

Plac(1) plasmid 153.7ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer (KaiA_BsaI_Mut_fwd)primer (KaiA_BsaI_Mut_rer)KOD plus-ver.2MilliQtotal
0.13 2.52.51.50.750.750.516.3725
KaiB (plasmid) 20.2ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer (GG2_RBS_KaiB_fwd)primer (KaiB_GG3_rer)KOD plus-ver.2MilliQtotal
12.52.51.50.750.750.515.525
KaiC (plasmid) 4.4ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer primer KOD plus-ver.2MilliQtotal
12.52.51.50.750.750.515.525
DT-1 (plasmid) 153.7ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer primer KOD plus-ver.2MilliQtotal
0.12.52.51.50.750.750.516.425
PKaiBC(PCR product) 16.3ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer primer KOD plus-ver.2MilliQtotal
0.72.52.51.50.750.750.515.825
SasA(PCR product) 21.6ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer primer KOD plus-ver.2MilliQtotal
0.52.52.51.50.750.750.51625
RpaA(PCR product) 15.4ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer primer KOD plus-ver.2MilliQtotal
0.62.52.51.50.750.750.515.925
RpaB(PCR product) 26.7ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer primer KOD plus-ver.2MilliQtotal
0.42.52.51.50.750.750.516.125
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C68°C--
5min10sec30sec30
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C68°C--
5min10sec30sec30


Mutatation PCR

No name

KaiABC(12.6pg/µL)Primer(Kai_BsaIMut_fwd)Primer(Kai_BsaIMut_rer)Prime STAR Max PremixMilliQtotal
1 11252250
Primer(Kai_BsaIMut_fwd)Primer(Kai_BsaIMut_rer)Prime STAR Max PremixMilliQtotal
11252350
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　65°C　72°C--
5min10sec　15sec　40sec　30

Colony PCR

Samplebase pair
9/1 pT181antisense(pSB1C3)400bp
9/1 pT181antisense-DT547bp
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
5m　30s　30s　30s　30

Electrophoresis

Kojima

LaneSample
1100bp ladder
29/1 pT181antisense(pSB1C3)
39/1 pT181antisense

Igku Sep3 Electrophoresis (N2).jpg

Electrophoresis

LaneSample
1 100bp ladder
2 attenuator EcoRI&SpeI
3 attenuator negative EcoRI&SpeI
4 attenuator XbaI&PstI
5 attenuator negative XbaI&PstI
6 antisense EcoRI&SpeI
7 antisense negative EcoRI&SpeI
8 100bp ladder
LaneSample
1 100bp ladder
2 antisense XbaI&PstI
3 antisense negative XbaI&PstI
4 apt EcoRI&SpeI
5 apt negative EcoRI&SpeI
6 DT EcoRI&XbaI
7 DT negative EcoRI&XbaI
8 100bp ladder

Igku Sep3 Electrophoresis(N3 2).jpg

LaneSample
1 100bp ladder
2 RBS-lysis1 EcoRI&SpeI
3 RBS-lysis1 negative
4 RBS-lysis2 EcoRI&SpeI
5 RBS-lysis2 negative
6 RBS-lysis3 EcoRI&SpeI
7 RBS-lysis3 negative
8 100bp ladder

Igku Sep3 Electrophoresis(N3 3).jpg

Gel Extraction

LaneDNAEnzyme
1 100bp ladder
3 pT181 attenuatorEcoRI&SpeI
4 pT181 attenuatorEcoRI&SpeI
6 pT181 attenuatorXbaI&PstI
7 pT181 attenuatorXbaI&PstI
9 pT181 antisenseEcoRI&SpeI
10 pT181 antisenseEcoRI&SpeI

Igku Sep3 Electrophoresis(N4 1).jpg

LaneDNAEnzyme
1 100bp ladder
2 pT181 antisenseXbaI&PstI
3 pT181 antisenseXbaI&PstI
5 apt12_1REcoRI&SpeI
6 apt12_1REcoRI&SpeI

Igku Sep3 Electrophoresis(N4 2).jpg

LaneDNAEnzyme
1 100bp ladder
2 DTEcoRI&XbaI
3 DTEcoRI&XbaI
5 RBS-lysis1EcoRI&SpeI
6 RBS-lysis1EcoRI&SpeI

Igku Sep3 Electrophoresis(N5 3).jpg

LaneDNAEnzyme
1 100bp ladder
2 RBS-lysis2EcoRI&SpeI
3 RBS-lysis2EcoRI&SpeI
5 RBS-lysis3EcoRI&SpeI
6 RBS-lysis3EcoRI&SpeI

Igku Sep3 Electrophoresis(N5 4).jpg

Nameconcentration[µg/mL]260/280260/230
pT181 attenuator(EcoRI&SpeI)10.0 1.86 0.39
pT181 attenuator(XbaI&PstI) 11.7 1.65 0.36
pT181 antisense(EcoRI&SpeI) 4.2 1.95 0.25
pT181 antisense(XbaI&PstI)6.6 1.78 0.33
tRNA 12_1R apt(EcoRI&SpeI)5.7 1.73 0.29
DT(EcoRI&XbaI) 16.6 1.99 0.76
RBS-lysis1(EcoRI&SpeI)8.8 1.65 0.32
RBS-lysis2(EcoRI&SpeI)13.4 1.61 0.48
RBS-lysis3(EcoRI&SpeI)10.2 2.19 0.51

Liquid Culture

No name

Samplemedium
8/16master plate7(DT)
8/21RBS-lysis1 　
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