Template:Kyoto/Notebook/Sep 26

From 2013.igem.org

(Difference between revisions)
(→PCR)
(→Colony PCR)
 
(17 intermediate revisions not shown)
Line 6: Line 6:
!Lane||Sample||Enzyme1||Enzyme2
!Lane||Sample||Enzyme1||Enzyme2
|-
|-
-
|1||GGA1-1|| ||  
+
|1||GGA1-1||--||--
|-
|-
-
|2||GGA1-2|| ||  
+
|2||GGA1-2||--||--
|-
|-
-
|3||GGA1-3|| ||  
+
|3||GGA1-3||--||--
|-
|-
-
|4||GGA1-4|| ||  
+
|4||GGA1-4||--||--
|-
|-
-
|5||GGA1-5|| ||  
+
|5||GGA1-5||--||--
|-
|-
-
|6||GGA1-6|| ||  
+
|6||GGA1-6||--||--
|-
|-
-
|7||GGA1-7|| ||  
+
|7||GGA1-7||--||--
|-
|-
-
|8||GGA1-8|| ||  
+
|8||GGA1-8||--||--
|-
|-
-
|9||1kbp ladder|| ||  
+
|9||1kbp ladder||--||--
|-
|-
-
|10||GGA2-1|| ||  
+
|10||GGA2-1||--||--
|-
|-
-
|11||GGA2-2|| ||  
+
|11||GGA2-2||--||--
|-
|-
-
|12||GGA2-3|| ||  
+
|12||GGA2-3||--||--
|-
|-
-
|13||GGA2-4|| ||  
+
|13||GGA2-4||--||--
|-
|-
-
|14||GGA2-5|| ||  
+
|14||GGA2-5||--||--
|-
|-
-
|15||GGA2-6|| ||  
+
|15||GGA2-6||--||--
|-
|-
-
|16||GGA2-7|| ||  
+
|16||GGA2-7||--||--
|-
|-
-
|17||GGA2-8|| ||  
+
|17||GGA2-8||--||--
|}
|}
[[File:Igku_0926_E1.jpg]]<br>
[[File:Igku_0926_E1.jpg]]<br>
-
</div>
 
{| class="wikitable"
{| class="wikitable"
!Lane||Sample||Enzyme1||Enzyme2
!Lane||Sample||Enzyme1||Enzyme2
|-
|-
-
|1||GGA3-1|| ||  
+
|1||GGA3-1||--||--
|-
|-
-
|2||GGA3-2|| ||  
+
|2||GGA3-2||--||--
|-
|-
-
|3||GGA3-3|| ||  
+
|3||GGA3-3||--||--
|-
|-
-
|4||1kbp ladder|| ||  
+
|4||1kbp ladder||--||--
|-
|-
-
|5||GGA3-4|| ||  
+
|5||GGA3-4||--||--
|-
|-
-
|6||GGA3-5|| ||  
+
|6||GGA3-5||--||--
|-
|-
-
|7||GGA3-6|| ||  
+
|7||GGA3-6||--||--
|-
|-
-
|8||GGA3-7|| ||  
+
|8||GGA3-7||--||--
|}
|}
-
</div>
 
{| class="wikitable"
{| class="wikitable"
!Lane||Sample||Enzyme1||Enzyme2
!Lane||Sample||Enzyme1||Enzyme2
|-
|-
-
|1||GGA3-8|| ||  
+
|1||GGA3-8||--||--
|-
|-
-
|2||GGA4-1|| ||  
+
|2||GGA4-1||--||--
|-
|-
-
|3||GGA4-2|| ||  
+
|3||GGA4-2||--||--
|-
|-
-
|4||GGA4-3|| ||  
+
|4||GGA4-3||--||--
|-
|-
-
|5||1kbp ladder|| ||  
+
|5||1kbp ladder||--||--
|-
|-
-
|6||GGA4-4|| ||  
+
|6||GGA4-4||--||--
|-
|-
-
|7||GGA4-5|| ||  
+
|7||GGA4-5||--||--
|-
|-
-
|8||GGA4-6|| ||  
+
|8||GGA4-6||--||--
|}
|}
[[File:Igku_0926_E2.jpg]]<br>
[[File:Igku_0926_E2.jpg]]<br>
-
</div>
+
 
{| class="wikitable"
{| class="wikitable"
!Lane||Sample||Enzyme1||Enzyme2
!Lane||Sample||Enzyme1||Enzyme2
|-
|-
-
|1||GGA4-7|| ||  
+
|1||GGA4-7||--||--
|-
|-
-
|2||GGA4-8|| ||  
+
|2||GGA4-8||--||--
|-
|-
-
|3||1kbp ladder|| ||  
+
|3||1kbp ladder||--||--
|-
|-
-
|4||GGA7-1|| ||  
+
|4||GGA7-1||--||--
|-
|-
-
|5||GGA16-1|| ||  
+
|5||GGA16-1||--||--
|-
|-
-
|6||NC(RBS-GFP-DT)|| ||  
+
|6||NC(RBS-GFP-DT)||--||--
|}
|}
[[File:Igku_0926_E3.jpg]]<br>
[[File:Igku_0926_E3.jpg]]<br>
Line 231: Line 229:
|}
|}
[[File:Igku_0926_E6.jpg]]<br>
[[File:Igku_0926_E6.jpg]]<br>
-
</div>
 
{| class="wikitable"
{| class="wikitable"
Line 263: Line 260:
|}
|}
[[File:Igku_0926_E7.jpg]]<br>
[[File:Igku_0926_E7.jpg]]<br>
-
</div>
 
{| class="wikitable"
{| class="wikitable"
Line 284: Line 280:
|8||GGA-3 (14)||--||--
|8||GGA-3 (14)||--||--
|}
|}
-
</div>
 
{| class="wikitable"
{| class="wikitable"
Line 370: Line 365:
<span class="author">Hirano</span>
<span class="author">Hirano</span>
{| class="wikitable"
{| class="wikitable"
-
!state||Vector||Inserter1||Inserter2||Inserter3||Inserter4
+
!state||Vector||Inserter1||Inserter2||Inserter3||Inserter4||Inserter5
|-
|-
-
|experiment||GGA2||Pcon-attenuator(E-1A)||Pcon-tetR-DT(1-2A)||Pcon-pT181 attenuator(1-SA)|
+
|experiment||GGA2||Pcon-attenuator(E-1A)||Pcon-tetR-DT(1-2A)||Ptet(2-S)||RBS-GFP-DT(EcoRI&XbaI)||
|-
|-
-
|experiment||GGA3||Pcon-tetap-DT(E-1A)||Pcon-tetR-DT(1-2A)||Pcon-GFP-DT(1-SA)||1.5 &micro;L||--||--||--||--||0.5 &micro;L||0.5 &micro;L||up to 5 &micro;L
+
|experiment||GGA3||Pcon-tetap-DT(E-1A)||Pcon-tetR-DT(1-2A)||Ptet(2-S)||RBS-GFP-DT(EcoRI&XbaI)||
|-
|-
-
|experiment||GGA4||Pcon-spi-DT(E-1A)||Pcon-tetR-DT(1-2A)||tet aptamer(1-3)||0.3 &micro;L||Pcon-tetR-DT(3-2A)||1.1 &micro;L||Ptet(2-5)|
+
|experiment||GGA4||Pcon-spi-DT(E-1A)||Pcon-tetR-DT(1-2A)||Ptet(2-S)||RBS-GFP-DT(EcoRI&XbaI)||
|-
|-
-
|experiment||GGA5||Pcon-antisense(E-1A)||Pcon-pT181 attenuator-DT(0-1A)||1.0 &micro;L||Pcon-tetR-DT(1-2A)||1.0 &micro;L||Ptet(2-5)||0.3&micro;L||--||--||0.5 &micro;L||0.5 &micro;L||up to 5 &micro;L
+
|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)||1.0 &micro;L||Pcon antisense-spinach-DT(1-2A)||0.7&micro;L||Pcon-pT 181 attenuator(2-SA)||2.1 &micro;L||--||--||--||--||0.5 &micro;L||0.5 &micro;L||up to 5 &micro;L
+
|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)||0.5 &micro;L||spinach-DT(1-2)||1.0 &micro;L||Pcon-GFP-DT(1-2)||0.8 &micro;L||--||--||--||--||0.5 &micro;L||0.5 &micro;L||up to 5 &micro;L
+
|experiment||GGA7||Pcon-tetR-DT(E-2A)||Ptet(2-S)||RBS-GFP-DT(EcoRI&XbaI)||||
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|37&deg;C||16&deg;C||50&deg;C||80&deg;C||--
 +
|-
 +
|3min||4min||5min||5min||25cycles
|}
|}
</div>
</div>
 +
 +
===RNA Extraction===
 +
<div class="experiment">
 +
<span class="author">Nakamoto</span>
 +
{| class="wikitable"
 +
!Sample
 +
|-
 +
|Pcon-Spinach-DT
 +
|-
 +
|Pcon-tetRaptamer-DT
 +
|-
 +
|Pcon-attenuator-DT
 +
|-
 +
|Spinach-DT
 +
|-
 +
|Pcon-antisense-Spinach-DT
 +
|}
 +
</div>
 +
 +
===Liquid Culture===
 +
<div class="experiment">
 +
<span class="author">Kozima</span>
 +
{| class="wikitable"
 +
!Sample
 +
|-
 +
|Pcon-attemuater-DT
 +
|-
 +
|Pcon-aptamer-DT
 +
|-
 +
|Pcon-spinach-DT
 +
|-
 +
|Pcon-RBS-tetR-DT
 +
|-
 +
|Pcon-antisense-spinach-DT(Master 1)
 +
|-
 +
|Pcon-antisense
 +
|}
 +
</div>
 +
 +
===Colony PCR===
 +
<div class="experiment">
 +
<span class="author">Nakamoto Kozima</span>
 +
{| class="wikitable"
 +
!Sample||base pair
 +
|-
 +
|9/25 9/19pSB1C3&9/3PT181 attenuator(1~3)||601bp
 +
|-
 +
|9/25 aptamer12_1R-DT&9/24Pcon-pT181 attenuator(1~4)||859bp
 +
|-
 +
|Pcon-attenuator-aptamer-DT(1)||859bp
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|　94&deg;C||　94&deg;C||　55&deg;C||　65&deg;C||--
 +
|-
 +
|5min||30sec　||30sec　||54sec　||30
 +
|}
 +
</div>
 +
 +
===Electrophoresis===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme1||Enzyme2
 +
|-
 +
|1||1kbp ladder||--||--
 +
|-
 +
|2||9/25 9/23 1C3&9/3attenuator||--||--
 +
|-
 +
|3||9/25 9/23 1C3&9/3attenuator||--||--
 +
|-
 +
|4||9/25 9/23 1C3&9/3attenuator||--||--
 +
|-
 +
|5||9/25aptamer-DT&9/24Pcon-attenuator||--||--
 +
|-
 +
|6||9/25aptamer-DT&9/24Pcon-attenuator||--||--
 +
|-
 +
|7||9/25aptamer-DT&9/24Pcon-attenuator||--||--
 +
|-
 +
|8||9/24 Pcon attenuator-aptamer-DT(1)||--||--
 +
|-
 +
|9||9/25aptamer-DT&9/24Pcon-attenuator||--||--
 +
|}
 +
[[File:Igku_0926_E10.jpg]]<br>
 +
</div>
 +
 +
===Plating===
 +
<div class="experiment">
 +
<span class="author">Hirano</span>
 +
</div>
 +
{| class="wikitable"
 +
!Sample||Use plate
 +
|-
 +
|9/26 GGA2 ||LB(CP)
 +
|-
 +
|9/26 GGA3 ||LB(CP)
 +
|-
 +
|9/26 GGA4||LB(CP)
 +
|-
 +
|9/26 GGA5 ||LB(CP)
 +
|-
 +
|9/26 GGA6 ||LB(CP)
 +
|-
 +
|9/26 GGA7 ||LB(CP)
 +
|-
 +
|9/26 GGA11 ||LB(CP)
 +
|-
 +
|9/26 GGA312||LB(CP)
 +
|-
 +
|9/26 GGA13 ||LB(CP)
 +
|-
 +
|9/26 GGA14||LB(CP)
 +
|-
 +
|9/26 GGA15 ||LB(CP)
 +
|-
 +
|9/26 GGA20||LB(CP)
 +
|-
 +
|9/26 GGA23 ||LB(CP)
 +
|-
 +
|9/26 GGA24||LB(CP)
 +
|-
 +
|9/26 Ptet+RBS-GFP-DT ||LB(CP)
 +
|}
 +
incubate 37&deg;C
 +
 +
===Restriction Enzyme Digestion===
 +
<div class="experiment">
 +
<span class="author">Kojima</span>
 +
{| class="wikitable"
 +
!||9/24 pSB1C3||EcoRI||SpeI||buffer||MilliQ||total
 +
|-
 +
|2cuts||11&micro;L||1&micro;L||1&micro;L||3&micro;L||14&micro;L||30&micro;L
 +
|-
 +
|NC||0.5&micro;L||0&micro;L||0&micro;L||1&micro;L||8.5&micro;L||10&micro;L
 +
|}
 +
 +
{| class="wikitable"
 +
!||9/24 Pcon-pT181attenuator-aptamer12-1R-DT||EcoRI||SpeI||buffer||MilliQ||total
 +
|-
 +
|2cuts||8.7&micro;L||1&micro;L||1&micro;L||3&micro;L||16.3&micro;L||30&micro;L
 +
|-
 +
|NC||0.4&micro;L||0&micro;L||0&micro;L||1&micro;L||8.6&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
!||9/24 pT181attenuator||EcoRI||SpeI||buffer||MilliQ||total
 +
|-
 +
|2cuts||3.1&micro;L||1&micro;L||1&micro;L||3&micro;L||21.9&micro;L||30&micro;L
 +
|-
 +
|NC||0.2&micro;L||0&micro;L||0&micro;L||1&micro;L||8.8&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
!||8/17 RBS-GFP-DT||EcoRI||BSA ||buffer||MilliQ||total
 +
|-
 +
|1cut||16.4&micro;L||1&micro;L||3&micro;L||3&micro;L||6.6&micro;L||30&micro;L
 +
|-
 +
|NC||0.4&micro;L||0&micro;L||1&micro;L||1&micro;L||7.6&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
!||9/17 pSB4K5||EcoRI||PstI||BSA ||buffer||MilliQ||total
 +
|-
 +
|2cuts||8.5&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||13.5&micro;L||30&micro;L
 +
|-
 +
|NC||0.4&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||7.6&micro;L||10&micro;L
 +
|}
 +
 +
{| class="wikitable"
 +
!||9/14 pSB4K5||EcoRI||PstI||BSA ||buffer||MilliQ||total
 +
|-
 +
|2cuts||7.3&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||14.7&micro;L||30&micro;L
 +
|-
 +
|NC||0.4&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||7.6&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
!||9/22 Pcon-pT181attenuator-DT||EcoRI||SpeI ||buffer||MilliQ||total
 +
|-
 +
|2cuts||5.4&micro;L||1&micro;L||1&micro;L||3&micro;L||19.6&micro;L||30&micro;L
 +
|-
 +
|NC||0.3&micro;L||0&micro;L||0&micro;L||1&micro;L||8.7&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
!||8/7 RBS-GFP-DT 1||XbaI||PstI||BSA ||buffer||MilliQ||total
 +
|-
 +
|2cuts||8.1&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||13.9&micro;L||30&micro;L
 +
|-
 +
|NC||0.4&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||7.6&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
!||9/26 RBS-GFP-DT 1||XbaI ||BSA ||buffer||MilliQ||total
 +
|-
 +
|1cut||8.1&micro;L||1&micro;L||3&micro;L||3&micro;L||14.9&micro;L||30&micro;L
 +
|}
 +
{| class="wikitable"
 +
!||9/16 P&lambda;-luxI||EcoRI||PstI||BSA ||buffer||MilliQ||total
 +
|-
 +
|2cuts||14.7&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||7.3&micro;L||30&micro;L
 +
|-
 +
|NC||0.7&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||7.3&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
!||9/21 Ptet||EcoRI||SpeI ||buffer||MilliQ||total
 +
|-
 +
|2cuts||14.2&micro;L||1&micro;L||1&micro;L||3&micro;L||10.8&micro;L||30&micro;L
 +
|-
 +
|NC||0.7&micro;L||0&micro;L||0&micro;L||1&micro;L||8.3&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
!||9/24 Pcon-pT181attenuator-aptamer12-1R-DT||XbaI||PstI||BSA ||buffer||MilliQ||total
 +
|-
 +
|2cuts||8.7&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||13.3&micro;L||30&micro;L
 +
|-
 +
|NC||0.4&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||7.6&micro;L||10&micro;L
 +
|}
 +
 +
</div>
 +
 +
===Electrophoresis===
 +
<div class="experiment">
 +
<span class="author">Kojima</span>
 +
{| class="wikitable"
 +
!Lane||Sample|| Enzyme1|| Enzyme2
 +
|-
 +
|1||pSB1C3||EcoRI||SpeI
 +
|-
 +
|2||pSB1C3||--||--
 +
|-
 +
|3||Pcon-pT181attenuator-aptamer12-1R-DT||EcoRI||SpeI
 +
|-
 +
|4||Pcon-pT181attenuator-aptamer12-1R-DT||--||--
 +
|-
 +
|5||1kb ladder||--||--
 +
|-
 +
|6||pT181attenuator||EcoRI||SpeI
 +
|-
 +
|7||pT181attenuator||--||--
 +
|-
 +
|8||RBS-GFP-DT||EcoRI||--
 +
|-
 +
|9||RBS-GFP-DT||--||--
 +
|}
 +
[[File:Igku Sep26 Electrophoresis(E11) 1.jpg]]<br>

Latest revision as of 18:26, 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----
LaneSampleEnzyme1Enzyme2
1GGA3-8----
2GGA4-1----
3GGA4-2----
4GGA4-3----
51kbp ladder----
6GGA4-4----
7GGA4-5----
8GGA4-6----

Igku 0926 E2.jpg


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

Igku 0926 E3.jpg

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

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

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

Nakamoto

Sample
Pcon-Spinach-DT
Pcon-tetRaptamer-DT
Pcon-attenuator-DT
Spinach-DT
Pcon-antisense-Spinach-DT

Liquid Culture

Kozima

Sample
Pcon-attemuater-DT
Pcon-aptamer-DT
Pcon-spinach-DT
Pcon-RBS-tetR-DT
Pcon-antisense-spinach-DT(Master 1)
Pcon-antisense

Colony PCR

Nakamoto Kozima

Samplebase pair
9/25 9/19pSB1C3&9/3PT181 attenuator(1~3)601bp
9/25 aptamer12_1R-DT&9/24Pcon-pT181 attenuator(1~4)859bp
Pcon-attenuator-aptamer-DT(1)859bp
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　65°C--
5min30sec　30sec　54sec　30

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
11kbp ladder----
29/25 9/23 1C3&9/3attenuator----
39/25 9/23 1C3&9/3attenuator----
49/25 9/23 1C3&9/3attenuator----
59/25aptamer-DT&9/24Pcon-attenuator----
69/25aptamer-DT&9/24Pcon-attenuator----
79/25aptamer-DT&9/24Pcon-attenuator----
89/24 Pcon attenuator-aptamer-DT(1)----
99/25aptamer-DT&9/24Pcon-attenuator----

Igku 0926 E10.jpg

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)

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----
Igku Sep26 Electrophoresis(E11) 1.jpg
Retrieved from "http://2013.igem.org/Template:Kyoto/Notebook/Sep_26"