Template:Kyoto/Notebook/Sep 2

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Contents

Sep 2

Colony PCR

Tatsui

Samplebase pair
9/1 RBS-lysis2-DT-(1)985
Ptet-RBS-lacZα-DT-(1)765
Plac-RBS-lacZα-DT-(1)765
Plac-RBS-lacZα-DT-(2)765
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min30cycles

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder
29/1 RBS-lysis2+DT -(1)
39/1 Ptet+RBS-lacZα-DT -(1)
49/1 Plac+RBS-lacZα-DT -(1)
59/1 Plac+RBS-lacZα-DT -(2)
6100bp ladder

Igku Sep2 ColonyPCR (N1-2).jpg

Restriction Enzyme Digestion

Nakamoto

pSB1C3-(1)EcoRISpeIXbaIPstIBufferBBufferDBSAMilliQtotal
2 cuts(EcoRI+SpeI)7µL1µL1µL0µL0µL3µL0µL0.3µL17.7µL30µL
NC(EcoRI+SpeI)0.3µL0µL0µL0µL0µL1µL0µL0.1µL8.6µL10µL
2 cuts(XbaI+PstI)7µL0µL0µL1µL1µL0µL3µL0.3µL17.7µL30µL
NC(XbaI+PstI)0.3µL0µL0µL0µL0µL0µL1µL0.1µL8.6µL10µL
8/21 tRMA-spinach(1)EcoRISpeIBufferBBSAMilliQtotal
2 cuts8µL1.0µL1.0µL3µL0.3µL17.6µL30µL
NC0.5µL0µL0µL1µL0.1µL8.4µL10µL
8/21 tetR aptamer12_1R-(1)EcoRISpeIBufferBBSAMilliQtotal
2 cuts6µL1.0µL1.0µL3µL0.3µL18.7µL30µL
NC0.4µL0µL0µL1µL0.1µL8.5µL10µL
8/21 pT181 attenuator-(2)EcoRISpeIXbaIPstIBufferBBufferDBSAMilliQtotal
2 cuts(EcoRI+SpeI)8µL1µL1µL0µL0µL3µL0µL0.3µL16.3µL30µL
NC(EcoRI+SpeI)0.5µL0µL0µL0µL0µL1µL0µL0.1µL8.4µL10µL
2 cuts(XbaI+PstI)8µL0µL0µL1µL1µL0µL3µL0.3µL16.3µL30µL
NC(XbaI+PstI)0.5µL0µL0µL0µL0µL0µL1µL0.1µL8.4µL10µL

Liquid Culture

Kojima

Samplemedium
tRNA-Spinach-1Plusgrow medium(+CP)
tetR aptamaer12_1R-1Plusgrow medium(+CP)
pT181 attenuator-1Plusgrow medium(+CP)　
pT181 antisense-1Plusgrow medium(+CP)

Master Plate

Kojima

NumberUse LB plate(+CP)
1tRNA Spinach-1
2tetR aptamaer12_1R
3tetR aptamaer12_P
4tetR aptamaer12_1M
5pT181 attenuator-2
6Fusion1 attenuator-1
7Fusion3m2 attenuator-1
8pT181 antisense-1
9Fusion1 antisense-1
10Fuaion6 antisense-1

Electrophoresis

Nakamoto & Tatsui

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2pSB1C3 -(1)EcoRISpeI
3pSB1C3 -(1)----
5pSB1C3 -(1)XbaIPstI
6pSB1C3 -(1)----
7pT181 attenuator (2)EcoRISpeI
8pT181 attenuator (2)----
9pT181 attenuator (2)XbaISpeI
10pT181 attenuator (2)----
11tetR-aptamer 12_1REcoRISpeI
12tetR-aptamer 12_1R----
13tRNA Spinach (1)EcoRISpeI
14100bp ladder----
15tRNA Spinach (1)----

Igku Sep2 Restriction Enzyme Digestion (N1-1).jpg

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder-
2pSB1C3(EcoRI+SpeI)EcoRI&SpeI
3
4----
58/24 tetR aptamer 12_1REcoRI&SpeI
6

Igku Sep1 Gel Extraction(N2-1).jpg
Igku Sep1 Gel Extraction(N2-2).jpg
No name

LaneDNAEnzyme
1100bp ladder-
2pT181 attenuator(2)(EcoRI+SpeI)EcoRI&SpeI
3
4----
5pT181 attenuator(2)(XbaI+PstI)EcoRI&SpeI
6
7----
8pT181 attenuator(2)(XbaI+PstI)EcoRI&SpeI
9
10----
11SpinachEcoRI&SpaI
12

Igku Sep1 Gel Extraction(N3-1).jpg
Igku Sep1 Gel Extraction(N3-2).jpg

Nameconcentration[µg/mL]260/280260/230
pSB1C3 (EcoRI&SpeI)3.02.180.31
pSB1C3 (XbaI&PstI)4.72.250.36
pT181 attenuator-(2)(EcoR&SpeI)8.62.740.01
pT181 attenuator-(2) (XbaI&PstI)16.52.460.03
Spinach (EcoRI&SpeI)2.82.980.27
tetR aptamer12_1R (EcoRI&SpeI)50.428.071.97

Colony PCR

No name

Samplebase pair
9/1 Spinach(pSB1C3)about 450
9/1 Spinach-DT596
9/1 RBS-lysis1-DT613
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s40s30cycles

Igku Sep2 ColonyPCR (N4).jpg

Samplebase pair
9/1 Ptet-RBS-lacZα-DT-2738
9/1 Ptet-RBS-lacZα-DT-3738
9/1 Ptet-RBS-lacZα-DT-4738
9/1 Ptet-RBS-lacZα-DT-5738
9/1 Ptet-RBS-lacZα-DT-6738
9/1 RBS-lysis2-DT-2985
9/1 RBS-lysis2-DT-3985
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min30cycles

Igku Sep3 ColonyPCR (N1).jpg

Master Plate

No name

NumberUse LB plate(+CP)
19/1 Spinach(pSB1C3)
29/1 Spinach-DT-(1)

Liquid Culture

No name

Samplemedium
pT181 attenuatorPlusgrow medium (+CP)
pT181 antisensePlusgrow medium (+CP)
SpinachPlusgrow medium (+CP)
  • incubated at 37 °C for 1 hour

Transformation

No name

NameSampleCompetent CellsTotalPlate
8/28 RBS-lysis3-DT3µL30µL33µLCP
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