Template:Kyoto/Notebook/Sep 24

From 2013.igem.org

(Difference between revisions)
(→Restriction Enzyme Digestion)
(→Ligation)
 
(23 intermediate revisions not shown)
Line 41: Line 41:
|17||9/20 Plac-aptamer12-1R-DT 4
|17||9/20 Plac-aptamer12-1R-DT 4
|}
|}
-
[[File:igkuxxxx.png]]<br>
+
[[File:igku_0924_E1.jpg]]<br>
</div>
</div>
Line 78: Line 78:
===Liquid Culture===
===Liquid Culture===
<div class="experiment">
<div class="experiment">
-
<span class="author">No name </span>
+
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
!Sample||medium
!Sample||medium
Line 85: Line 85:
|}
|}
</div>
</div>
 +
 +
===Restriction Enzyme Digestion(PCR product)===
 +
<div class="experiment">
 +
<span class="author">Hirano</span>
 +
Concentrate PCR product by evaporator until volume become less than 1&micro;L, add all sulution and pipetting. </div>
 +
{| class="wikitable"
 +
!||10*cut smart buffer||BsaI-HF||MilliQ||total
 +
|-
 +
|9/22 Pcon-pT181 attenuator (E-0) ||2.0 &micro;L ||0.3 &micro;L ||17 &micro;L ||19.3&micro;L
 +
|-
 +
|9/22 Pcon-pT181 attenuator (2-3A) ||2.0 &micro;L ||0.3 &micro;L ||17 &micro;L ||19.3&micro;L
 +
|-
 +
|9/22 spinach-DT(3-2) ||2.0 &micro;L ||0.3 &micro;L ||17 &micro;L ||19.3&micro;L
 +
|-
 +
|9/22 Ptet-pT181 antisense (1-3) ||2.0 &micro;L ||0.3 &micro;L ||17 &micro;L ||19.3&micro;L
 +
|-
 +
|9/22 Pcon-tet aptamer-DT (E-1A) ||2.0 &micro;L ||0.3 &micro;L ||17 &micro;L ||19.3&micro;L
 +
|-
 +
|9/22 Pcon-spinach-DT (E-1A) ||2.0 &micro;L ||0.3 &micro;L ||17 &micro;L ||19.3&micro;L
 +
|-
 +
|9/22 Pcon-pT181 attenuator (E-1A) ||2.0 &micro;L ||0.3 &micro;L ||17 &micro;L ||19.3&micro;L
 +
|-
 +
|9/22 DT (1-3) ||2.0 &micro;L ||0.3 &micro;L ||17 &micro;L ||19.3&micro;L
 +
|-
 +
|9/22 tet aptamer-DT (1-3) ||2.0 &micro;L ||0.3 &micro;L ||17 &micro;L ||19.3&micro;L
 +
|-
 +
|9/22 Pcon-pT181 attenuator (1-SA) ||2.0 &micro;L ||0.3 &micro;L ||17 &micro;L ||19.3&micro;L
 +
|-
 +
|9/22 Pcon-RBS-tetR-DT (1-2A) ||2.0 &micro;L ||0.3 &micro;L ||17 &micro;L ||19.3&micro;L
 +
|-
 +
|9/22 Pcon-RBS-tetR-DT (3-2A) ||2.0 &micro;L ||0.3 &micro;L ||17 &micro;L ||19.3&micro;L
 +
|-
 +
|9/22 Pcon-RBS-tetR-DT (E-2A) ||2.0 &micro;L ||0.3 &micro;L ||17 &micro;L ||19.3&micro;L
 +
|-
 +
|9/22 Pcon-RBS-GFP-DT (1-2A) ||2.0 &micro;L ||0.3 &micro;L ||17 &micro;L ||19.3&micro;L
 +
|-
 +
|9/22 Pcon-RBS-GFP-DT (2-SA) ||2.0 &micro;L ||0.3 &micro;L ||17 &micro;L ||19.3&micro;L
 +
|-
 +
|9/23 Pcon-pT181 attenuator-DT (E-1A) ||2.0 &micro;L ||0.3 &micro;L ||17 &micro;L ||19.3&micro;L
 +
|-
 +
|9/23 Pcon-pT181 attenuator-DT (0-1A) ||2.0 &micro;L ||0.3 &micro;L ||17 &micro;L ||19.3&micro;L
 +
|-
 +
|9/23 Pcon-pT181 antisense-spinach-DT (1-2A) ||2.0 &micro;L ||0.3 &micro;L ||17 &micro;L ||19.3&micro;L
 +
|-
 +
|9/23 Pcon-pT181 antisense (1-2A) ||2.0 &micro;L ||0.3 &micro;L ||17 &micro;L ||19.3&micro;L
 +
|-
 +
|9/23 Ptet-1 (2-S) ||2.0 &micro;L ||0.3 &micro;L ||17 &micro;L ||19.3&micro;L
 +
|-
 +
|9/23 Plac-2 (E-1A) ||2.0 &micro;L ||0.3 &micro;L ||17 &micro;L ||19.3&micro;L
 +
|}
 +
Incubate 37&deg;C 12hour</div>
 +
 +
===Column Refining===
 +
<div class="experiment">
 +
<span class="author">Kojima</span>
 +
{| class="wikitable"
 +
!Name||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|8/22 Pcon-pT181 attenuator(E-0)||7.8||1.70||0.03
 +
|-
 +
|8/22 Pcon-pT181 attenuator(2-SA)||17.3||1.93||0.03
 +
|-
 +
|8/22 spinach-DT (3-2)||22.4||1.93||0.09
 +
|-
 +
|8/22 Ptet-PT181 anisense (1-3)||18.3||1.58||0.14
 +
|-
 +
|8/22 Pcon-aptamer12_1R-DT (E-1A)||8.2||2.10||0.05
 +
|-
 +
|8/22 Pcon-spinach-DT(E-1A)||5.6||0.91||0.05
 +
|-
 +
|8/22 Pcon-pT181 attenuator(E-1A)||18.5||1.80||0.12
 +
|-
 +
|8/22 DT(1-3)||7.3||1.13||0.04
 +
|-
 +
|8/22 tetR aptamer 12_1R-DT(1-3)||14.6||1.74||0.06
 +
|-
 +
|8/22 Pcon-pT181 attenuator(1-SA)||7.9||1.57||0.04
 +
|-
 +
|8/22 Pcon-RBS-tetR-DT(1-2Ⓐ)||41.6||1.81||0.25
 +
|-
 +
|8/22 Pcon-RBS-tetR-DT(3-2Ⓐ)||30.2||1.75||0.15
 +
|-
 +
|8/22 Pcon-RBS-tetR-DT(E-2Ⓐ)||36.9||1.75||0.56
 +
|-
 +
|8/22 Pcon-RBS-GFP-DT(1-SⒶ)||43.5||1.83||0.33
 +
|-
 +
|8/22 Pcon-RBS-GFP-DT(2-SⒶ)||64.6||1.82||0.30
 +
|-
 +
|8/23 Pcon-pT181 attenuator-DT (E-1A)||31.2||1.83||0.09
 +
|-
 +
|8/23 Pcon-pT181 attenuator-DT (0-1A)||64.5||1.80||0.43
 +
|-
 +
|8/23 Pcon-pT181 antisense-spinach-DT (1-2A)||36.4||1.88||0.21
 +
|-
 +
|8/23 Pcon-pT181 antisense-spinach-DT (E-1A)||27.9||1.92||0.16
 +
|-
 +
|8/23 Ptet(1)(2-S)||37.0||1.91||0.16
 +
|-
 +
|9/23 Plac-2(E-1A) ||21.2||1.82||0.13
 +
|}
===Transformation===
===Transformation===
Line 111: Line 211:
</div>
</div>
-
=== Digestion===
+
===Restriction Enzyme Digestion===
<div class="experiment">
<div class="experiment">
<span class="author">Kojima </span>
<span class="author">Kojima </span>
Line 157: Line 257:
|7|| pSB1C3 ||--||--
|7|| pSB1C3 ||--||--
|}<br>
|}<br>
-
[[File:xxx]]<br>
+
[[File:Igku_0924_E2.jpg]]<br>
<br>
<br>
</div>
</div>
 +
===Miniprep===
===Miniprep===
<div class="experiment">
<div class="experiment">
Line 200: Line 301:
|12|| pSB1C3||EcoRI||SpeI  
|12|| pSB1C3||EcoRI||SpeI  
|}
|}
-
[[File:Igkuxxx.jpg]]<br>
+
[[File:Igku_0924_G1.jpg]]<br>
-
[[File:Igku Axxxjpg]]<br>
+
[[File:Igku_0924_G2.jpg]]<br>
{| class="wikitable"
{| class="wikitable"
Line 322: Line 423:
|17||9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 8
|17||9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 8
|}
|}
-
[[File:igkuxxxx.png]]<br>
+
[[File:igku_0924_E3.jpg]]<br>
</div>
</div>
Line 356: Line 457:
|14||--
|14||--
|}
|}
-
[[File:igkuxxxx.png]]<br>
+
[[File:igku_0924_E4.jpg]]<br>
</div>
</div>
Line 411: Line 512:
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
|-
|-
-
|||9/22 aptamer12-1R-DT(EcoRI&XbaI) ||1.4 &micro;L||9/24 Pcon-pT181attenuator(EcoRI&SpeI)||26 &micro;L||3.5 &micro;L
+
|experiment||9/22 aptamer12-1R-DT(EcoRI&XbaI) ||1.4 &micro;L||9/24 Pcon-pT181attenuator(EcoRI&SpeI)||26 &micro;L||3.5 &micro;L
|-
|-
-
|||9/4 Ptet(SpeI&PstI)||2.0 &micro;L||9/21 RBS-GFP-DT(XbaI&PstI)||9.0 &micro;L||3.5 &micro;L
+
|experiment||9/4 Ptet(SpeI&PstI)||2.0 &micro;L||9/21 RBS-GFP-DT(XbaI&PstI)||9.0 &micro;L||3.5 &micro;L
|}
|}
</div>
</div>
Line 508: Line 609:
!Lane||Sample||Enzyme1||Enzyme2
!Lane||Sample||Enzyme1||Enzyme2
|-
|-
-
|1kb ladder||--||--
+
|1||1kb ladder||--||--
|-
|-
|2||9/24 aptamer12-1R-DT||EcoRI||XbaI
|2||9/24 aptamer12-1R-DT||EcoRI||XbaI
Line 520: Line 621:
|6||9/24 RBS-GFP-DT ||EcoRI||SpeI
|6||9/24 RBS-GFP-DT ||EcoRI||SpeI
|-
|-
-
|7| 9/24 RBS-GFP-DT ||-- ||--
+
|7||9/24 RBS-GFP-DT ||-- ||--
|-
|-
|8||9/24 Pcon-RBS-GFP-DT ||EcoRI||XbaI
|8||9/24 Pcon-RBS-GFP-DT ||EcoRI||XbaI
Line 536: Line 637:
|13||9/24 RBS-GFP-DT ||--||--
|13||9/24 RBS-GFP-DT ||--||--
|-
|-
-
|14||1kb ladder
+
|14||1kb ladder||--||--
|}
|}
-
[[File:Igkuxxxx.jpg]]<br>
+
[[File:Igku_0924_E5.jpg]]<br>
===Gel Extraction===
===Gel Extraction===
Line 571: Line 672:
|12||RBS-GFP-DT||EcoRI||SpeI
|12||RBS-GFP-DT||EcoRI||SpeI
|}
|}
-
[[File:Igku xxxbefore.jpg]]<br>
+
[[File:Igku_0924_G3.jpg]]<br>
-
[[File:Igkuxxx1-2.jpg]]<br>
+
[[File:Igku_0924_G4.jpg]]<br>
{| class="wikitable"
{| class="wikitable"
Line 601: Line 702:
|12||RBS-GFP-DT||XbaI||PstI
|12||RBS-GFP-DT||XbaI||PstI
|}
|}
-
[[File:Igkuxxxxpg]]<br>
+
[[File:Igku_0924_G5.jpg]]<br>
-
[[File:Igkuxxxx2.jpg]]<br>
+
[[File:Igku_0924_G6.jpg]]<br>
<br>
<br>
Line 628: Line 729:
|8||1kb ladder
|8||1kb ladder
|}
|}
-
[[File:Igkuxxxx.jpg]]<br>
 
<br>
<br>
Line 651: Line 751:
|8||1kb ladder
|8||1kb ladder
|}
|}
-
[[File:Igkuxxxx.jpg]]<br>
+
[[File:Igku_0924_E6.jpg]]<br>
<br>
<br>
Line 674: Line 774:
|8|| Pcon-pT181attenuator-aptamer12-1R-DT 2
|8|| Pcon-pT181attenuator-aptamer12-1R-DT 2
|}
|}
-
[[File:Igkuxxxx.jpg]]<br>
 
<br>
<br>
Line 696: Line 795:
|-
|-
|}
|}
-
[[File:Igkuxxxx.jpg]]<br>
+
[[File:Igku_0924_E7.jpg]]<br>
 +
 
 +
===Ligation===
 +
 +
<div class="experiment">
 +
<span class="author">Hirano</span>
 +
{| class="wikitable"
 +
!state||colspan="2"|Vector||colspan="2"|Inserter1||colspan="2"|Inserter2||colspan="2"|Inserter3||colspan="2"|Inserter4||(NEB)T4ligase||(NEB)Buffer||milliQ||total
 +
|-
 +
|experiment||RBS-GFP-DT(pSB1C3)||1&micro;L||tetR-aptamer12-1R-DT(1-3)||1 &micro;L||Ptet(2-S)||0.3&micro;L||Pcon-tetR-aptamer12-1R-DT(3-2A)||1.1&micro;L||Plac(E-1A)||0.5&micro;L||0.5&micro;L||0.5&micro;L||0.2 &micro;L||5&micro;L
 +
|-
 +
|experiment||RBS-GFP-DT(pSB1C3)||1&micro;L||Pcon-pT181attenuator(E-1A)||1 &micro;L||Pcon-tetR-aptamer12-1R-DT(1-2A)||1&micro;L||Ptet(2-S)||0.3&micro;L||-- ||-- ||0.5&micro;L||0.5&micro;L||0.7 &micro;L||5&micro;L
 +
|-
 +
|experiment||RBS-GFP-DT(pSB1C3)||1&micro;L||Pcon-tetRaptamer12-1R-DT(E-1A)||2&micro;L||Pcon-tetRaptamer12-1R-DT(1-2A)||1&micro;L||Ptet(2-S)||0.3&micro;L||--||--||0.5&micro;L||0.5&micro;L||0 &micro;L||5.3&micro;L
 +
|-
 +
|experiment||RBS-GFP-DT(pSB1C3)||1&micro;L||Pcon-spinach-DT(E-1A)||3&micro;L||Pcon-tetRaptamer12-1R-DT(1-2A)||1&micro;L||Ptet(2-S)||0.3&micro;L||--||--||0.5&micro;L||0.5&micro;L||0 &micro;L||6.3&micro;L
 +
|-
 +
|experiment||RBS-GFP-DT(pSB1C3)||1&micro;L||Pcon-pT181antisense-DT(E-1A)||0.5&micro;L||DT(1-3)||2&micro;L||Ptet(2-S)||0.3&micro;L||Pcon-tetRaptamer12-1R-DT(3-2A)||1.1&micro;L||0.5&micro;L||0.5&micro;L||0.1 &micro;L||5&micro;L
 +
|-
 +
|experiment||RBS-GFP-DT(pSB1C3)||1&micro;L||Pcon-pT181attenuator(E-1A)||1&micro;L||tetRaptamer12-1R-DT(1-3)||1&micro;L||Ptet(2-S)||0.3&micro;L||Pcon-tetRaptamer12-1R-DT(3-2A)||1.1&micro;L||0.5&micro;L||0.5&micro;L||0&micro;L||5.4&micro;L
 +
|-
 +
|experiment||RBS-GFP-DT(pSB1C3)||1&micro;L||Ptet(2-S)||0.3&micro;L||Pcon-tetRaptamer12-1R-DT(E-2A)||1&micro;L||--||--||--||--||0.5&micro;L||0.5&micro;L||1.7&micro;L||5&micro;L
 +
|-
 +
|experiment||RBS-GFP-DT(pSB1C3)||1&micro;L||Pcon-GFP-DT(2-SA)||0.5&micro;L||Pcon-tetRaptamer12-1R-DT(E-2A)|1&micro;L||--||--||--||--||--||0.5&micro;L||0.5&micro;L||1.5&micro;L||5&micro;L
 +
|-
 +
|experiment||RBS-GFP-DT(pSB1C3)||1&micro;L||Pcon-tetRaptamer12-1R-DT(E-1A)||2&micro;L||Pcon-pT181attenuator(1-SA)||2.1&micro;L||--||--||--||--||0.5&micro;L||0.5&micro;L||0&micro;L||6.1&micro;L
 +
|-
 +
|experiment||RBS-GFP-DT(pSB1C3)||1&micro;L||Pcon-pT181attenuator-DT(E-1A)||1&micro;L||Pcon-pT181attenuator(1-SA)||2.1&micro;L||--||--||--||--||0.5&micro;L||0.5&micro;L||0&micro;L||5.1&micro;L
 +
|-
 +
|experiment||RBS-GFP-DT(pSB1C3)||1&micro;L||Pcon-tetR12-1R-DT(E-1A)||2&micro;L||Pcon-GFP-DT(2-SA)||0.5&micro;L||spinach-DT(3-2)||1&micro;L||Ptet-pT181antisense(1-3)||1&micro;L||0.5&micro;L||0.5&micro;L||0&micro;L||6.5&micro;L
 +
|-
 +
|experiment||RBS-GFP-DT(pSB1C3)||1&micro;L||Pcon-tetRaptamer12-1R-DT(E-1A)||2&micro;L||Pcon-pT181antisense(1-2A)||0.7&micro;L||Pcon-pT181attenuator(2-SA)||2.1&micro;L||--||--||0.5&micro;L||0.5&micro;L||0&micro;L||6.8&micro;L
 +
|-
 +
|experiment||RBS-GFP-DT(pSB1C3)||1&micro;L||Pcon-tetRaptamer12-1R-DT(E-1A)||2&micro;L||Pcon-pT181attenuator(2-SA)||2.1&micro;L||Spinach-DT(3-2)||1&micro;L||Ptet-pT181antisense(1-3)||1&micro;L||0.5&micro;L||0.5&micro;L||0&micro;L||8.1&micro;L
 +
|}
 +
<br>Concentrate total (more than 5&micro;L) by evaporater until volume become less than 5 &micro;L<br>
 +
</div>
 +
 
 +
===PCR===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||F primer||R primer||KOD-plus||MilliQ||total
 +
|-
 +
|Pcon antisense(1C3-GGE-fwd,1A2-GG2-rev)||1&micro;L||2.5&micro;L||2.5&micro;L||1.5&micro;L||0.75&micro;L||0.75&micro;L||0.5&micro;L||15.5||25&micro;L
 +
|-
 +
|DT-(1C3-GG1-fwd,1C3-GG2-rev)||1&micro;L||2.5&micro;L||2.5&micro;L||1.5&micro;L||0.75&micro;L||0.75&micro;L||0.5&micro;L||15.5||25&micro;L
 +
|-
 +
|Spinach-DT-(1C3-GG1-fwd,1C3-GG2-rev)||1&micro;L||2.5&micro;L||2.5&micro;L||1.5&micro;L||0.75&micro;L||0.75&micro;L||0.5&micro;L||15.5||25&micro;L
 +
|-
 +
|Pcon-antisense-spinach-DT(1C3-GGE-fwd,1A2-GG1-rev) ||1&micro;L||2.5&micro;L||2.5&micro;L||1.5&micro;L||0.75&micro;L||0.75&micro;L||0.5&micro;L||15.5||25&micro;L
 +
|-
 +
|Ptet(1C3-GGE-fwd,1C3-GG0-rev) ||1&micro;L||2.5&micro;L||2.5&micro;L||1.5&micro;L||0.75&micro;L||0.75&micro;L||0.5&micro;L||15.5||25&micro;L
 +
|-
 +
|Pcon attenuator(1C3-GGE-fwd,1C3-GG0-rev) ||1&micro;L||2.5&micro;L||2.5&micro;L||1.5&micro;L||0.75&micro;L||0.75&micro;L||0.5&micro;L||15.5||25&micro;L
 +
|-
 +
|DT(1C3-GG2-fwd,1C3-GG1-rev)||1&micro;L||2.5&micro;L||2.5&micro;L||1.5&micro;L||0.75&micro;L||0.75&micro;L||0.5&micro;L||15.5||25&micro;L
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||98&deg;C||57&deg;C||68&deg;C||--
 +
|-
 +
|2min||10sec||30sec||36sec||30
 +
|}
 +
</div>

Latest revision as of 18:22, 27 September 2013

Contents

Sep 24

Electrophoresis

No name

LaneSample
19/20 Pcon-pT181aptamer12-1R-DT 1
29/20 Pcon-pT181aptamer12-1R-DT 2
39/20 Pcon-pT181aptamer12-1R-DT 3
49/20 Pcon-pT181aptamer12-1R-DT 4
59/20 Pcon-pT181aptamer12-1R-DT 5
69/20 Pcon-pT181aptamer12-1R-DT 6
79/20 Pcon-pT181aptamer12-1R-DT 7
89/20 Pcon-pT181aptamer12-1R-DT 8
9100bp ladder
109/22 Pcon-pT181attenuator-aptamer12-1R-DT 1
119/22 Pcon-pT181attenuator-aptamer12-1R-DT 2
129/22 Pcon-pT181attenuator-aptamer12-1R-DT 3
139/22 Pcon-pT181attenuator-aptamer12-1R-DT 4
149/20 Plac-aptamer12-1R-DT 1
159/20 Plac-aptamer12-1R-DT 2
169/20 Plac-aptamer12-1R-DT 3
179/20 Plac-aptamer12-1R-DT 4

Igku 0924 E1.jpg

Transformation

No name

NameSampleCompetent CellsTotalPlate
9/23 pSB1C3-pT181attenuator 2 µL20 µL 22 µLLB (+CP)
RBS-GFP-DT+Pcon-pT181attenuator 2 µL20 µL 22 µLLB (+CP)
9/23 Plac-+spinach-DT2 µL20 µL 22 µLLB (+Amp)
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 2 µL20 µL 22 µLLB (+Amp)
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 2 µL20 µL 22 µLLB (+Amp)
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 2 µL20 µL 22 µLLB (+Amp)
9/23 Pcon-pT181antisense-DT 2 µL20 µL 22 µLLB (+CP)
9/23 Pcon-pT181attenuator-RBS-GFP-DT 2 µL20 µL 22 µLLB (+CP)
9/23 Pcon-pT181attenuator-aptamer12-1R-DT 2 µL20 µL 22 µLLB (+CP)
PUC192 µL20 µL 22 µLLB (+Amp)
Ptet-RBS-lacZα-DT 2 µL20 µL 22 µLLB (+CP)
pSB1C32 µL20 µL 22 µLLB (+CP)

Liquid Culture

No name

Samplemedium
aptamer12-1R-DT(Master plate) LB(+CP)

Restriction Enzyme Digestion(PCR product)

Hirano

Concentrate PCR product by evaporator until volume become less than 1µL, add all sulution and pipetting.
10*cut smart bufferBsaI-HFMilliQtotal
9/22 Pcon-pT181 attenuator (E-0) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-pT181 attenuator (2-3A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 spinach-DT(3-2) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Ptet-pT181 antisense (1-3) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-tet aptamer-DT (E-1A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-spinach-DT (E-1A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-pT181 attenuator (E-1A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 DT (1-3) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 tet aptamer-DT (1-3) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-pT181 attenuator (1-SA) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-RBS-tetR-DT (1-2A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-RBS-tetR-DT (3-2A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-RBS-tetR-DT (E-2A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-RBS-GFP-DT (1-2A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-RBS-GFP-DT (2-SA) 2.0 µL 0.3 µL 17 µL 19.3µL
9/23 Pcon-pT181 attenuator-DT (E-1A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/23 Pcon-pT181 attenuator-DT (0-1A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/23 Pcon-pT181 antisense-spinach-DT (1-2A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/23 Pcon-pT181 antisense (1-2A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/23 Ptet-1 (2-S) 2.0 µL 0.3 µL 17 µL 19.3µL
9/23 Plac-2 (E-1A) 2.0 µL 0.3 µL 17 µL 19.3µL

Incubate 37°C 12hour</div>

Column Refining

Kojima

Nameconcentration[µg/mL]260/280260/230
8/22 Pcon-pT181 attenuator(E-0)7.81.700.03
8/22 Pcon-pT181 attenuator(2-SA)17.31.930.03
8/22 spinach-DT (3-2)22.41.930.09
8/22 Ptet-PT181 anisense (1-3)18.31.580.14
8/22 Pcon-aptamer12_1R-DT (E-1A)8.22.100.05
8/22 Pcon-spinach-DT(E-1A)5.60.910.05
8/22 Pcon-pT181 attenuator(E-1A)18.51.800.12
8/22 DT(1-3)7.31.130.04
8/22 tetR aptamer 12_1R-DT(1-3)14.61.740.06
8/22 Pcon-pT181 attenuator(1-SA)7.91.570.04
8/22 Pcon-RBS-tetR-DT(1-2Ⓐ)41.61.810.25
8/22 Pcon-RBS-tetR-DT(3-2Ⓐ)30.21.750.15
8/22 Pcon-RBS-tetR-DT(E-2Ⓐ)36.91.750.56
8/22 Pcon-RBS-GFP-DT(1-SⒶ)43.51.830.33
8/22 Pcon-RBS-GFP-DT(2-SⒶ)64.61.820.30
8/23 Pcon-pT181 attenuator-DT (E-1A)31.21.830.09
8/23 Pcon-pT181 attenuator-DT (0-1A)64.51.800.43
8/23 Pcon-pT181 antisense-spinach-DT (1-2A)36.41.880.21
8/23 Pcon-pT181 antisense-spinach-DT (E-1A)27.91.920.16
8/23 Ptet(1)(2-S)37.01.910.16
9/23 Plac-2(E-1A) 21.21.820.13

Transformation

No name

NameSampleCompetent CellsTotalPlate
9/23 Pcon-attenuator-aptamer12-1R-DT 2 µL20 µL 22 µLLB (+CP)
9/23 pT181attenuator(1C3) 2 µL20 µL 22 µLLB (+CP)
9/23 Pcon-pT181antisense-DT2 µL20 µL 22 µLLB (+CP)
9/23 Pcon-pT181attenuator-RBS--GFP-DT 2 µL20 µL 22 µLLB (+CP)

Liquid Culture

No name

Samplemedium
pSB1C3 (Master plate-1) LB(+CP)

Restriction Enzyme Digestion

Kojima

8/21 pT181attenuator (330 µg/mL) XbaIPstIbuffer BSAMilliQtotal
2 cuts6.1113315.930
NC0.300117.710
9/22 Pcon-pT181attenuator (368.3 µg/mL) EcoRISpeIbuffer BSAMilliQtotal
2 cuts5.4113019.630
NC0.300108.710
9/16 pSB1C3EcoRISpeIbuffer BSAMilliQtotal
2 cuts1811307.030
NC0.900108.110

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
11kb ladder--
2pT181attenuator XbaIPstI
3 pT181attenuator ----
4Pcon-pT181attenuator EcoRISpeI
5 Pcon-pT181attenuator ----
6pSB1C3 EcoRISpeI
7 pSB1C3 ----

Igku 0924 E2.jpg

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
9/24 aptamer12-1R-DT144.71.031.21

Gel Extraction

Kojima

LaneDNAEnzyme1Enzyme2
1100kb ladder----
2pT181attenuatorXbaIPstI
3 pT181attenuatorXbaIPstI
4 pT181attenuatorXbaIPstI
5------
6Pcon-pT181attenuatorEcoRISpeI
7 Pcon-pT181attenuatorEcoRISpeI
8 Pcon-pT181attenuatorEcoRISpeI
9------
10pSB1C3EcoRISpeI
11 pSB1C3EcoRISpeI
12 pSB1C3EcoRISpeI

Igku 0924 G1.jpg
Igku 0924 G2.jpg

Nameconcentration[µg/mL]260/280260/230
pT181attenuator(XbaI&PstI)10.1 2.02 0.43
Pcon-pT181attenuator(EcoRI&SpeI)10.3 1.81 0.32
pSB1C3(EcoRI&SpeI)10.02.810.02

Colony PCR

Nakamoto

Samplebase pair
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 11414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 21414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 31414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 41414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 51414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 61414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 71414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 81414
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 11631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 21631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 31631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 41631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 51631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 61631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 71631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 81631
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 12042
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 22042
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 32042
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 42042
9/23 Pcon-pT181antisense-DT 1508
9/23 Pcon-pT181antisense-DT 2508
9/23 Pcon-pT181antisense-DT 3508
9/23 Pcon-pT181antisense-DT 4508
9/23 Pcon-pT181antisense-DT 5508
9/23 Pcon-pT181antisense-DT 6508
9/23 Pcon-pT181antisense-DT 7508
9/23 Pcon-pT181antisense-DT 8508
PreDenatureDenatureAnnealingExtensioncycle
94 °C94 °C55 °C68 °C--
5 min30 sec30 sec2min30 cycles
LaneSample
19/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 1
29/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 2
39/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 3
49/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 4
59/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 5
69/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 6
79/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 7
89/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 8
91kb ladder
109/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 1
119/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 2
129/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 3
139/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 4
149/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 5
159/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 6
169/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 7
179/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 8

Igku 0924 E3.jpg

LaneSample
19/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 1
29/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 2
39/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 3
49/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 4
59/23 Pcon-pT181antisense-DT 1
69/23 Pcon-pT181antisense-DT 2
71kb ladder
89/23 Pcon-pT181antisense-DT 3
99/23 Pcon-pT181antisense-DT 4
109/23 Pcon-pT181antisense-DT 5
119/23 Pcon-pT181antisense-DT 6
129/23 Pcon-pT181antisense-DT 7
139/23 Pcon-pT181antisense-DT 8
14--

Igku 0924 E4.jpg
</div>

Restriction Enzyme Digestion

Nakamoto

9/24 aptamer12-1R-DT EcoRIXbaI bufferBSAMilliQtotal
2cuts17.5µL1.0µL 1.0µL 3.0µL 3.0µL 4.5µL 30µL
NC0.9µL 0µL 0µL 1.0µL 1.0µL 7.1µL 10µL
9/20 PtetSpeIPstI bufferBSAMilliQtotal
2cuts9.5µL1.0µL 1.0µL 3.0µL 0µL 15.5µL 30µL
NC0.5µL 0µL 0µL 1.0µL 0µL 8.5µL 10µL
9/22 RBS-GFP-DTEcoRI SpeI bufferBSAMilliQtotal
2cuts3.8µL1.0µL 1.0µL 3.0µL 0µL 21.2µL 30µL
NC0.2µL 0µL 0µL 1.0µL 0µL 8.8µL 10µL
9/22 Pcon-RBS-GFP-DTEcoRI XbaI bufferBSAMilliQtotal
2cuts2.4µL1.0µL 1.0µL 3.0µL 3.0µL 19.6µL 30µL
NC0.1µL 0µL 0µL 1.0µL 1.0µL 7.9µL 10µL
9/22 Pcon-pT181attenuator 1 EcoRI XbaI bufferBSAMilliQtotal
2cuts5.4µL1.0µL 1.0µL 3.0µL 3.0µL 16.6µL 30µL
NC0.3µL 0µL 0µL 1.0µL 1.0µL 7.7µL 10µL
9/22 RBS-GFP-DTXbaI PstI bufferBSAMilliQtotal
2cuts3.8µL1.0µL 1.0µL 3.0µL 3.0µL 18.2µL 30µL
NC0.2µL 0µL 0µL 1.0µL 1.0µL 7.8µL 10µL

Ligation

Nakamoto

stateVectorInserterLigation High ver.2
experiment9/22 aptamer12-1R-DT(EcoRI&XbaI) 1.4 µL9/24 Pcon-pT181attenuator(EcoRI&SpeI)26 µL3.5 µL
experiment9/4 Ptet(SpeI&PstI)2.0 µL9/21 RBS-GFP-DT(XbaI&PstI)9.0 µL3.5 µL
  • incubated at 37 °C for 1 hour.

colony PCR

Kojima

Samplebase pair
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 52042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 62042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 72042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 82042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 92042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 102042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 112042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 122042bp
9/23 Pcon-pT181antisense-DT 9508bp
9/23 Pcon-pT181antisense-DT 10508bp
9/23 Pcon-pT181antisense-DT 11508bp
9/23 Pcon-pT181antisense-DT 12508bp
9/23 Pcon-pT181antisense-DT 13508bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 11527bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 21527bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 31527bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 41527bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 1859bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 2859bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 3859bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 4859bp
9/24 pT181attenuator(pSB1C3) 1390bp
9/24 pT181attenuator(pSB1C3) 2390bp
9/24 pT181attenuator(pSB1C3) 3390bp
9/24 pT181attenuator(pSB1C3) 4390bp

Tatsui

PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
5min30s　30s　2min30cycle

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
pSB1C3183.9　1.91　1.59
Pcon-pT181attenuator-aptamer12-1R-DT231.71.822.35
Plac-aptamer12-1R-DT208.91.981.79

Electrophoresis

Nakamoto

LaneSampleEnzyme1Enzyme2
11kb ladder----
29/24 aptamer12-1R-DTEcoRIXbaI
3 9/24 aptamer12-1R-DT -- --
49/24 Ptet SpeI PstI
59/24 Ptet ----
69/24 RBS-GFP-DT EcoRISpeI
79/24 RBS-GFP-DT -- --
89/24 Pcon-RBS-GFP-DT EcoRIXbaI
89/24 Pcon-RBS-GFP-DT ----
99/24 Pcon-RBS-GFP-DT ----
109/24 Pcon-pT181attenuator 1 EcoRIXbaI
119/24 Pcon-pT181attenuator 1 ----
129/24 RBS-GFP-DT XbaIPstI
139/24 RBS-GFP-DT ----
141kb ladder----

Igku 0924 E5.jpg

Gel Extraction

No name 1

LaneDNAEnzyme1Enzyme2
11kb ladder----
2aptamer12-1R-DTEcoRIXbaI
3aptamer12-1R-DTEcoRIXbaI
4aptamer12-1R-DTEcoRIXbaI
5-- -- --
6PtetSpeIPstI
7PtetSpeIPstI
8PtetSpeIPstI
9------
10RBS-GFP-DTEcoRISpeI
11RBS-GFP-DTEcoRISpeI
12RBS-GFP-DTEcoRISpeI

Igku 0924 G3.jpg
Igku 0924 G4.jpg

LaneDNAEnzyme1Enzyme2
11kb ladder----
2Pcon-RBS-GFP-DTEcoRIXbaI
3Pcon-RBS-GFP-DTEcoRIXbaI
4Pcon-RBS-GFP-DTEcoRIXbaI
5------
6Pcon-pT181attenuatorEcoRIXbaI
7Pcon-pT181attenuatorEcoRIXbaI
8Pcon-pT181attenuatorEcoRIXbaI
9------
10RBS-GFP-DTXbaIPstI
11RBS-GFP-DTXbaIPstI
12RBS-GFP-DTXbaIPstI

Igku 0924 G5.jpg
Igku 0924 G6.jpg

Electrophoresis

No name
1

LaneSample
11kb ladder
2Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 5
3Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 6
4Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 7
5Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 8
6Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 9
7Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 10
81kb ladder


2

LaneSample
11kb ladder
2Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 11
3Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 12
4Pcon-pT181attenuator-RBS-GFP-DT 1
5Pcon-pT181attenuator-RBS-GFP-DT 2
6Pcon-pT181attenuator-RBS-GFP-DT 3
7Pcon-pT181attenuator-RBS-GFP-DT 4
81kb ladder

Igku 0924 E6.jpg


3

LaneSample
1Pcon-pT181antisense-DT 9
2 Pcon-pT181antisense-DT 10
3 Pcon-pT181antisense-DT 11
4 Pcon-pT181antisense-DT 12
5 Pcon-pT181antisense-DT 13
6100bp ladder
7Pcon-pT181attenuator-aptamer12-1R-DT 1
8 Pcon-pT181attenuator-aptamer12-1R-DT 2


4

LaneSample
1Pcon-pT181attenuator-aptamer12-1R-DT 3
2 Pcon-pT181attenuator-aptamer12-1R-DT 4
3 100bp ladder
4 pT181attenuator(pSB1C3) 1
5 pT181attenuator(pSB1C3) 2
6 pT181attenuator(pSB1C3) 3
7 pT181attenuator(pSB1C3) 4

Igku 0924 E7.jpg

Ligation

Hirano

stateVectorInserter1Inserter2Inserter3Inserter4(NEB)T4ligase(NEB)BuffermilliQtotal
experimentRBS-GFP-DT(pSB1C3)1µLtetR-aptamer12-1R-DT(1-3)1 µLPtet(2-S)0.3µLPcon-tetR-aptamer12-1R-DT(3-2A)1.1µLPlac(E-1A)0.5µL0.5µL0.5µL0.2 µL5µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-pT181attenuator(E-1A)1 µLPcon-tetR-aptamer12-1R-DT(1-2A)1µLPtet(2-S)0.3µL-- -- 0.5µL0.5µL0.7 µL5µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-tetRaptamer12-1R-DT(E-1A)2µLPcon-tetRaptamer12-1R-DT(1-2A)1µLPtet(2-S)0.3µL----0.5µL0.5µL0 µL5.3µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-spinach-DT(E-1A)3µLPcon-tetRaptamer12-1R-DT(1-2A)1µLPtet(2-S)0.3µL----0.5µL0.5µL0 µL6.3µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-pT181antisense-DT(E-1A)0.5µLDT(1-3)2µLPtet(2-S)0.3µLPcon-tetRaptamer12-1R-DT(3-2A)1.1µL0.5µL0.5µL0.1 µL5µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-pT181attenuator(E-1A)1µLtetRaptamer12-1R-DT(1-3)1µLPtet(2-S)0.3µLPcon-tetRaptamer12-1R-DT(3-2A)1.1µL0.5µL0.5µL0µL5.4µL
experimentRBS-GFP-DT(pSB1C3)1µLPtet(2-S)0.3µLPcon-tetRaptamer12-1R-DT(E-2A)1µL--------0.5µL0.5µL1.7µL5µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-GFP-DT(2-SA)0.5µL1µL----------0.5µL0.5µL1.5µL5µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-tetRaptamer12-1R-DT(E-1A)2µLPcon-pT181attenuator(1-SA)2.1µL--------0.5µL0.5µL0µL6.1µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-pT181attenuator-DT(E-1A)1µLPcon-pT181attenuator(1-SA)2.1µL--------0.5µL0.5µL0µL5.1µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-tetR12-1R-DT(E-1A)2µLPcon-GFP-DT(2-SA)0.5µLspinach-DT(3-2)1µLPtet-pT181antisense(1-3)1µL0.5µL0.5µL0µL6.5µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-tetRaptamer12-1R-DT(E-1A)2µLPcon-pT181antisense(1-2A)0.7µLPcon-pT181attenuator(2-SA)2.1µL----0.5µL0.5µL0µL6.8µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-tetRaptamer12-1R-DT(E-1A)2µLPcon-pT181attenuator(2-SA)2.1µLSpinach-DT(3-2)1µLPtet-pT181antisense(1-3)1µL0.5µL0.5µL0µL8.1µL


Concentrate total (more than 5µL) by evaporater until volume become less than 5 µL

PCR

No name

genome DNAKOD plus10x bufferdNTPMgSO4F primerR primerKOD-plusMilliQtotal
Pcon antisense(1C3-GGE-fwd,1A2-GG2-rev)1µL2.5µL2.5µL1.5µL0.75µL0.75µL0.5µL15.525µL
DT-(1C3-GG1-fwd,1C3-GG2-rev)1µL2.5µL2.5µL1.5µL0.75µL0.75µL0.5µL15.525µL
Spinach-DT-(1C3-GG1-fwd,1C3-GG2-rev)1µL2.5µL2.5µL1.5µL0.75µL0.75µL0.5µL15.525µL
Pcon-antisense-spinach-DT(1C3-GGE-fwd,1A2-GG1-rev) 1µL2.5µL2.5µL1.5µL0.75µL0.75µL0.5µL15.525µL
Ptet(1C3-GGE-fwd,1C3-GG0-rev) 1µL2.5µL2.5µL1.5µL0.75µL0.75µL0.5µL15.525µL
Pcon attenuator(1C3-GGE-fwd,1C3-GG0-rev) 1µL2.5µL2.5µL1.5µL0.75µL0.75µL0.5µL15.525µL
DT(1C3-GG2-fwd,1C3-GG1-rev)1µL2.5µL2.5µL1.5µL0.75µL0.75µL0.5µL15.525µL
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10sec30sec36sec30
Retrieved from "http://2013.igem.org/Template:Kyoto/Notebook/Sep_24"