Template:Kyoto/Notebook/Aug 27

From 2013.igem.org

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Revision as of 16:37, 25 September 2013

Contents

Aug 27

Restriction Enzyme Digestion

No name

8/21 pT181 attenuator-1(330µg/mL)EcoRISpeI10x BufferB100x BSAMilliQtotal
2 cuts3.0µL0.5µL0.5µL3.0µL0.3µL22.7µL30µL
NC0.6µL0µL0µL1µL0.1µL8.3µL10µL
8/17 DT-1(188µg/mL)EcoRIXbaI10x BufferB100x BSAMilliQtotal
2 cuts3.0µL0.5µL0.5µL3.0µL0.3µL22.7µL30µL
NC1.0µl0µL0µL1µL0.1µL7.9µL10µL
8/20 Pcon-RBS-luxR-DT-2(344µg/mL)EcoRISpeI10x BufferB100x BSAMilliQtotal
2 cuts2.9µL0.5µL0.5µL3.0µL0.3µL22.8µL30µL
NC0.6µl0µL0µL1µL0.1µL8.3µL10µL

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder----
28/21 pT181 attenuatorEcoISpeI
38/21 pT181 atteniator----
48/17 DT-1EcoRIXbaI
58/17 DT-1----
68/20 Pcon-RBS-luxR-DT-2EcoISpeI
78/20 Pcon-RBS-luxR-DT-2----
8100bp ladder----

Igku Aug27 Electrophoresis(N1)-1.jpg

Colony PCR

Kojima and Yoshida

Samplebase pair
8/26 Pλ-luxI(1)
8/26 Pλ-luxI(2)
NC
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s3min30cycles

Liquid Culture

No name

Samplemedium
8/26 Pλ-RBS-luxI-DT-1Plusgrow medium (+Amp)
8/26 Pλ-RBS-luxI-DT-2Plusgrow medium (+Amp)　

Restriction Enzyme Digestion

No name

8/21 pT181 attenuator-1(330µg/mL)EcoRISpeI10x BufferB100x BSAMilliQtotal
2 cuts3.0µL0.5µL0.5µL3.0µL0.3µL22.7µL30µL
NC0.6µL0µL0µL1µL0.1µL8.3µL10µL
8/17 DT-1(188µg/mL)EcoRIXbaI10x BufferB100x BSAMilliQtotal
2 cuts3.0µL0.5µL0.5µL3.0µL0.3µL22.7µL30µL
NC1.0µl0µL0µL1µL0.1µL7.9µL10µL
8/20 Pcon-RBS-luxR-DT-2(344µg/mL)EcoRISpeI10x BufferB100x BSAMilliQtotal
2 cuts2.9µL0.5µL0.5µL3.0µL0.3µL22.8µL30µL
NC0.6µl0µL0µL1µL0.1µL8.3µL10µL

Electrophoresis

Hirano

LaneSampleEnzyme1Enzyme2
100bp ladder----
1pT181 attenuatorEcoR1SpeI
2pT181 attenuator----
3DT-(1)EcoRIXbaI
4DT-(1)----
5Pcon-RBS-luxR-DT-(2)EcoRISpeI
6Pcon-RBS-luxR-DT-(2)----
100bp ladder----

Igku Aug27 Electrophoresis(N2)-2.jpg

Gel Extraction

Inoue

LaneDNAEnzyme
1100bpladder--
2pT181 attenuator-1EcoRI&SpeI
3pT181 attenuator-1EcoRI&SpeI
4DT-1EcoRI&XbaI
5DT-1EcoRI&XbaI
6Pcon-RBS-luxR-DT-2EcoRI&SpeI
7Pcon-RBS-luxR-DT-2EcoRI&SpeI
8100bpladder--

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Nameconcentration[µg/mL]260/280260/230
pT181 attenuator-1(EcoRI&SpeI)4.21.620.29
DT-1(EcoRI&XbaI)171.880.88

Transformation

Kojima and Hirano

NameSampleCompetent CellsTotalPlate
8/26 tetR aptamer 12_1R-DT1µL10µL11µLCP
8/26 pT181 attenuator-DT1µL10µL11µLCP
8/26 pT181 antisense-DT1µL10µL11µLCP
8/26 Spinach-DT1µL10µL11µLCP
8/26 Plac-pT181 attenuator1µL10µL11µLCP
8/26 Pcon-pT181 attenuator1µL10µL11µLAmp
8/26 Plac-pT181 antisense1µL10µL11µLCP
8/26 Pcon-pT181 antisense1µL10µL11µLAmp

Liquid culture

No name

Samplemedium
8/16 PluxPlusgrow medium(+CP)

Genome PCR

Yoshdia and Nakagawa

genome DNAKOD plus10x bufferdNTPMgSO4SasA_fwd primerSasA_rev primerMilliQtotal
0.50.52.52.51.50.750.751625
genome DNAKOD plus10x bufferdNTPMgSO4RpaA_fwd primerRpaA_rev primerMilliQtotal
0.50.52.52.51.50.750.751625
genome DNAKOD plus10x bufferdNTPMgSO4RpaB_fwd primerRpaB_rev primerMilliQtotal
0.50.52.52.51.50.750.751625
genome DNAKOD plus10x bufferdNTPMgSO4PkaiBC_fwd primerPkaiBC_rev primerMilliQtotal
0.50.52.52.51.50.750.751625
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C50°C68°C--
2 min10 sec30 sec38 sec30 cycles

Ligation

じっけんしゃ

stateVectorInserter
experiment8/27 DT (EcoRI & XbaI)2.88/21 RBS-lysis3 (EcoRI & SpeI)
experiment8/27 DT (EcoRI & XbaI)2.88/27 pT181 attenuator (EcoRI & SpeI)
  • Samples were evaporeted used evaporator into about 3 µL.
sampleMilliQLigation Hightotal
343.510.5
  • incubate overnight at 4 °C
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