Template:Kyoto/Notebook/Sep 26

From 2013.igem.org

(Difference between revisions)
(Ligasion(Golden Gate Assenbly))
(Ligasion(Golden Gate Assenbly))
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|experiment||GGA5||Pcon-antisense(E-1A)||DT(1-3)||Pcon-tetR-DT(3-2A)||Ptet(2-S)||RBS-GFP-DT(EcoRI&XbaI)
|experiment||GGA5||Pcon-antisense(E-1A)||DT(1-3)||Pcon-tetR-DT(3-2A)||Ptet(2-S)||RBS-GFP-DT(EcoRI&XbaI)
|-
|-
-
|experiment||GGA6||Pcon-attenuator(E-1A)||tetap-DT(1-3)||Pcon-tetR-DT(3-2A)||Ptet(2-S)||RBS-GFP-DT(EcoRI&XbaI)||
+
|experiment||GGA6||Pcon-attenuator(E-1A)||tetap-DT(1-3)||Pcon-tetR-DT(3-2A)||Ptet(2-S)||RBS-GFP-DT(EcoRI&XbaI)
|-
|-
|experiment||GGA7||Pcon-tetR-DT(E-2A)||Ptet(2-S)||RBS-GFP-DT(EcoRI&XbaI)||||
|experiment||GGA7||Pcon-tetR-DT(E-2A)||Ptet(2-S)||RBS-GFP-DT(EcoRI&XbaI)||||
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|37°C||16°C||50°C||80°C||--
 +
|-
 +
|3min||4min||5min||5min||25cycles
|}
|}
</div>
</div>

Revision as of 07:36, 27 September 2013

Contents

Sep 26

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1GGA1-1
2GGA1-2
3GGA1-3
4GGA1-4
5GGA1-5
6GGA1-6
7GGA1-7
8GGA1-8
91kbp ladder
10GGA2-1
11GGA2-2
12GGA2-3
13GGA2-4
14GGA2-5
15GGA2-6
16GGA2-7
17GGA2-8

Igku 0926 E1.jpg

LaneSampleEnzyme1Enzyme2
1GGA3-1
2GGA3-2
3GGA3-3
41kbp ladder
5GGA3-4
6GGA3-5
7GGA3-6
8GGA3-7

</div>

LaneSampleEnzyme1Enzyme2
1GGA3-8
2GGA4-1
3GGA4-2
4GGA4-3
51kbp ladder
6GGA4-4
7GGA4-5
8GGA4-6

Igku 0926 E2.jpg
</div>

LaneSampleEnzyme1Enzyme2
1GGA4-7
2GGA4-8
31kbp ladder
4GGA7-1
5GGA16-1
6NC(RBS-GFP-DT)

Igku 0926 E3.jpg
</div>

Ligasion(Golden Gate Assenbly)

No name

stateVectorInserter1Inserter2Inserter3Inserter4Inserter5(NEB)T4 ligase(NEB)10*T4 ligaseMilliQ
experimentRBS-GFP-DT1.0 µLPcon-pT181 antisense(E-2A)0.6µLDT(2-1)0.5µLPcon-pT181 attenuator(1-SA)2.1 µL--------0.5 µL0.5 µLup to 5 µL
experimentpSB1C3(EcoRI&SpeI)1.8 µLPcon-pT181 antisense(E-2A)0.9 µLDT(2-1)0.5 µLPcon-GFP-DT(1-SA)1.5 µL--------0.5 µL0.5 µLup to 5 µL
experimentRBS-GFP-DT1.0 µLPtet(E-0)0.7µLPcon pT181 attenuator(0-1A)0.3µLtet aptamer(1-3)0.3 µLPcon-tetR-DT(3-2A)1.1 µLPtet(2-5)0.3 µL0.5 µL0.5 µLup to 5 µL
experimentRBS-GFP-DT1.0 µLPtet(E-0)0.7 µLPcon-pT181 attenuator-DT(0-1A)1.0 µLPcon-tetR-DT(1-2A)1.0 µLPtet(2-5)0.3µL----0.5 µL0.5 µLup to 5 µL
experimentRBS-GFP-DT1.0 µLPcon-pT181 attenuator-tetR-DT(E-1A)1.0 µLPcon antisense-spinach-DT(1-2A)0.7µLPcon-pT 181 attenuator(2-SA)2.1 µL--------0.5 µL0.5 µLup to 5 µL
experimentRBS-GFP-DT1.0 µLPcon antisense(E-1A)0.5 µLspinach-DT(1-2)1.0 µLPcon-GFP-DT(1-2)0.8 µL--------0.5 µL0.5 µLup to 5 µL
experimentpSB1C3(EcoRI&SpeI)1.8 µLPcon-pT181 antisense(E-1A)0.8µLspinach-DT(1-2)1.0µLPcon-GFP-DT(2-SA)0.8 µL--------0.5 µL0.5 µLup to 5 µL
experimentpSB1C3(EcoRI&SpeI)1.8 µLPcon-tet aptamer-DT(E-1A)3.0 µLPtet-pT181 antisense(1-3)1.5 µLspinach-DT(3-2)1.5 µLPcon-GFP-DT(2-5A)0.8 µL----0.5 µL0.5 µLup to 5 µL

Liquid Culture

No name

Samplemedium
Pcon-GFP-DTAmp
RBS-GFP-DTCP
Pcon-pT181 attenuator-DTAmp
Pcon-tet aptamer-DTAmp
Pcon-spinach-DTAmp
Pcon-pT181 antisense-spinach-DTAmp
spinach-DTCP

BSAI Digestion

No name

DNAMilliQBSAI-HF10*cut smarttotal
Pcon7 µL10.7 µL0.3 µL2 µL20 µL
RBS-luxR-DT10 µL7 µL0.3 µL2 µL20 µL

Igku 0926 E4.jpg

Colony PCR

Tatsui

Samplebase pair
GGA-1 (9~16)3177
GGA-2 (9~16)3189
GGA-3 (9~21)2989
GGA-4 (9~16)2939
GGA-6 (1)3359
GGA-16(2~4)2939
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s3min30s30cycles

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1GGA-1 (9)----
2GGA-1 (10)----
3GGA-1 (11)----
4GGA-1 (12)----
5GGA-1 (13)----
6GGA-1 (14)----
7GGA-1 (15)----
8GGA-1 (16)----
91kbp ladder----
10GGA-2 (9)----
11GGA-2 (10)----
12GGA-2 (11)----
13GGA-2 (12)----
14GGA-2 (13)----
15GGA-2 (14)----
16GGA-2 (15)----
17GGA-2 (16)----

Igku 0926 E6.jpg

LaneSampleEnzyme1Enzyme2
1GGA-4 (9)----
2GGA-4 (10)----
3GGA-4 (11)----
4GGA-4 (12)----
5GGA-4 (13)----
6GGA-4 (14)----
7GGA-4 (15)----
8GGA-4 (16)----
91kbp ladder----
10GGA-6 (1)----
11GGA-16 (2)----
12GGA-16 (3)----
13GGA-16 (4)----

Igku 0926 E7.jpg
</div>

LaneSampleEnzyme1Enzyme2
1GGA-3 (9)----
2GGA-3 (10)----
3GGA-3 (11)----
4GGA-3 (12)----
51kbp ladder----
6GGA-3 (13)----
7GGA-3 (15)----
8GGA-3 (14)----

</div>

LaneSampleEnzyme1Enzyme2
1GGA-3 (17)----
2GGA-3 (18)----
3GGA-3 (19)----
41kbp ladder----
5GGA-3 (20)----
6GGA-3 (21)----
7GGA-3 (16)----

Igku 0926 E8.jpg
</div>

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1RBS-GFP-DT(RNA)----
21kbp ladder----
3Pcon-RBS-GFP-DT(RNA)----

Igku 0926 E9.jpg

Liquid Culture

Hirano

Samplemedium
Pcon-GFP-DT--
RBS-GFP-DT--
Pcon-tet aptamer-DT--
Pcon-spinach-DT--
Pcon-pT181 antisense-spinach-DT--
spinach-DT--

Colony PCR

Hirano

Samplebase pair
9/25 Pcon-pH181 attenuator+RBS-GFP-DT-1--
9/25 Pcon-pH181 attenuator+RBS-GFP-DT-2--
9/25 Pcon-pH181 attenuator+RBS-GFP-DT-3--
9/25 Pcon-pH181 attenuator+RBS-GFP-DT-4--
9/24 Ptet-RBS-GFP-DT-1--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min40s30cycles

Ligasion(Golden Gate Assenbly)

Hirano

stateVectorInserter1Inserter2Inserter3Inserter4Inserter5
experimentGGA2Pcon-attenuator(E-1A)Pcon-tetR-DT(1-2A)Ptet(2-S)RBS-GFP-DT(EcoRI&XbaI)
experimentGGA3Pcon-tetap-DT(E-1A)Pcon-tetR-DT(1-2A)Ptet(2-S)RBS-GFP-DT(EcoRI&XbaI)
experimentGGA4Pcon-spi-DT(E-1A)Pcon-tetR-DT(1-2A)Ptet(2-S)RBS-GFP-DT(EcoRI&XbaI)
experimentGGA5Pcon-antisense(E-1A)DT(1-3)Pcon-tetR-DT(3-2A)Ptet(2-S)RBS-GFP-DT(EcoRI&XbaI)
experimentGGA6Pcon-attenuator(E-1A)tetap-DT(1-3)Pcon-tetR-DT(3-2A)Ptet(2-S)RBS-GFP-DT(EcoRI&XbaI)
experimentGGA7Pcon-tetR-DT(E-2A)Ptet(2-S)RBS-GFP-DT(EcoRI&XbaI)
PreDenatureDenatureAnnealingExtensioncycle
37°C16°C50°C80°C--
3min4min5min5min25cycles

RNA Extraction

Liquid Culture

Colony PCR

Plating

Hirano

SampleUse plate
9/26 GGA2 LB(CP)
9/26 GGA3 LB(CP)
9/26 GGA4LB(CP)
9/26 GGA5 LB(CP)
9/26 GGA6 LB(CP)
9/26 GGA7 LB(CP)
9/26 GGA11 LB(CP)
9/26 GGA312LB(CP)
9/26 GGA13 LB(CP)
9/26 GGA14LB(CP)
9/26 GGA15 LB(CP)
9/26 GGA20LB(CP)
9/26 GGA23 LB(CP)
9/26 GGA24LB(CP)
9/26 Ptet+RBS-GFP-DT LB(CP)

</div> incubate 37°C

Restriction Enzyme Digestion

Kojima

9/24 pSB1C3EcoRISpeIbufferMilliQtotal
2cuts11µL1µL1µL3µL14µL30µL
NC0.5µL0µL0µL1µL8.5µL10µL
9/24 Pcon-pT181attenuator-aptamer12-1R-DTEcoRISpeIbufferMilliQtotal
2cuts8.7µL1µL1µL3µL16.3µL30µL
NC0.4µL0µL0µL1µL8.6µL10µL
9/24 pT181attenuatorEcoRISpeIbufferMilliQtotal
2cuts3.1µL1µL1µL3µL21.9µL30µL
NC0.2µL0µL0µL1µL8.8µL10µL
8/17 RBS-GFP-DTEcoRIBSA bufferMilliQtotal
1cut16.4µL1µL3µL3µL6.6µL30µL
NC0.4µL0µL1µL1µL7.6µL10µL
9/17 pSB4K5EcoRIPstIBSA bufferMilliQtotal
2cuts8.5µL1µL1µL3µL3µL13.5µL30µL
NC0.4µL0µL0µL1µL1µL7.6µL10µL
9/14 pSB4K5EcoRIPstIBSA bufferMilliQtotal
2cuts7.3µL1µL1µL3µL3µL14.7µL30µL
NC0.4µL0µL0µL1µL1µL7.6µL10µL
9/22 Pcon-pT181attenuator-DTEcoRISpeI bufferMilliQtotal
2cuts5.4µL1µL1µL3µL19.6µL30µL
NC0.3µL0µL0µL1µL8.7µL10µL
8/7 RBS-GFP-DT 1XbaIPstIBSA bufferMilliQtotal
2cuts8.1µL1µL1µL3µL3µL13.9µL30µL
NC0.4µL0µL0µL1µL1µL7.6µL10µL
9/26 RBS-GFP-DT 1XbaI BSA bufferMilliQtotal
1cut8.1µL1µL3µL3µL14.9µL30µL
9/16 Pλ-luxIEcoRIPstIBSA bufferMilliQtotal
2cuts14.7µL1µL1µL3µL3µL7.3µL30µL
NC0.7µL0µL0µL1µL1µL7.3µL10µL
9/21 PtetEcoRISpeI bufferMilliQtotal
2cuts14.2µL1µL1µL3µL10.8µL30µL
NC0.7µL0µL0µL1µL8.3µL10µL
9/24 Pcon-pT181attenuator-aptamer12-1R-DTXbaIPstIBSA bufferMilliQtotal
2cuts8.7µL1µL1µL3µL3µL13.3µL30µL
NC0.4µL0µL0µL1µL1µL7.6µL10µL

Electrophoresis

Kojima

LaneSample Enzyme1 Enzyme2
1pSB1C3EcoRISpeI
2pSB1C3----
3Pcon-pT181attenuator-aptamer12-1R-DTEcoRISpeI
4Pcon-pT181attenuator-aptamer12-1R-DT----
51kb ladder----
6pT181attenuatorEcoRISpeI
7pT181attenuator----
8RBS-GFP-DTEcoRI--
9RBS-GFP-DT----
File:IgkuxxxxBa 1.jpg