Template:Kyoto/Notebook/Sep 2

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Contents

Sep 2

Liquid Culture

Hirano

Samplemedium
9/1 entA-(Master Plate)-14mL SOB(+Km)

37°C

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

File:Igku sep2electrophoresis1.png

Restriction Enzyme Digestion

Nakamoto

pSB1C3-(1)EcoRISpeIXbaIPstIBufferBBufferDBSAMilliQtotal
2 cuts(E+S)7µL1µL1µL0µL0µL3µL0µL0.3µL17.7µL30µL
NC(E+S)0.3µL0µL0µL0µL0µL1µL0µL0.1µL8.6µL10µL
2 cut(X+P)s7µL0µL0µL1µL1µL0µL3µL0.3µL17.7µL30µL
NC(X+P)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(E+S)8µL1µL1µL0µL0µL3µL0µL0.3µL16.3µL30µL
NC(E+S)0.5µL0µL0µL0µL0µL1µL0µL0.1µL8.4µL10µL
2 cut(X+P)s8µL0µL0µL1µL1µL0µL3µL0.3µL16.3µL30µL
NC(X+P)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

Gel Extraction

EDTA Solution

Hirano

Colony PCR

Hirano

Master Plate

Hirano

Liquid Culture

Hirano

Colony PCR

Hirano

Transformation

Hirano
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