Template:Kyoto/Notebook/Sep 2

From 2013.igem.org

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[[File:Igku Sep2 Restriction Enzyme Digestion (N1-1).jpg]]<br>
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Revision as of 12:53, 24 September 2013

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

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

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

No name

LaneDNAEnzyme
1100bp ladder-
2pSB1C3(E+S)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)(E+S)EcoRI&SpeI
3
4----
5pT181 attenuator(2)(X+P)EcoRI&SpeI
6
7----
8pT181 attenuator(2)(X+P)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

File:Igku sep2electrophoresis1.png

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

File:Igku Sep2electrophoresis2.png

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)
  • 37&dig:C ihour incubate

Transformation

No name

NameSampleCompetent CellsTotalPlate
8/28 RBS-lysis3-DT3µL30µL33µLCP
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