Template:Kyoto/Notebook/Sep 22

From 2013.igem.org

(Difference between revisions)
(→Gel Extraction)
(→PCR)
 
(32 intermediate revisions not shown)
Line 15: Line 15:
|}
|}
</div>
</div>
 +
[[File:Igku_0903_E1.jpg]]<br>
 +
[[File:Igku_0922_E2.jpg]]<br>
 +
 +
===Electrophoresis===
 +
<div class="experiment">
 +
<span class="author">Nakamoto</span>
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1||Pcon-apt12_1R-DT(E-1A)
 +
|-
 +
|2||Pcon-spinach-DT(E-1A)
 +
|-
 +
|3||Pcon-pT181 attenuator(E-1A)
 +
|-
 +
|4||DT(1-3)
 +
|-
 +
|5||tetR aptamer 12_1R-DT(1-3)
 +
|-
 +
|6||100bp ladder
 +
|-
 +
|7||Pcon-pT181 attenuator(1-SA)
 +
|-
 +
|8||spinach-DT(2-1)
 +
|-
 +
|9||Pcon-pT181 attenuator(E-0)
 +
|-
 +
|10||Pcon-pT181 attenuator(2-SA)
 +
|-
 +
|11||spinach-DT(3-2)
 +
|-
 +
|12||Ptet-PT181 anisense
 +
|}
 +
[[File:Igku_0922_E3.jpg]]<br>
 +
[[File:Igku_0922_E4.jpg]]<br>
===Miniprep===
===Miniprep===
Line 51: Line 86:
|5min||30s||30s||2min10s||30cycles
|5min||30s||30s||2min10s||30cycles
|}
|}
 +
[[File:igku_0922_E5.jpg]]<br>
 +
[[File:igku_0922_E6.jpg]]<br>
===Gel Extraction===
===Gel Extraction===
Line 95: Line 132:
|}
|}
</div>
</div>
 +
[[File:Igku 0922 G1.jpg]]<br>
 +
[[File:Igku 0922 G2.jpg]]<br>
 +
[[File:Igku 0922 G3.jpg]]<br>
 +
[[File:Igku 0922 G4.jpg]]<br>
-
===digestion===
+
===Restriction Enzyme Digestion===
<div class="experiment">
<div class="experiment">
<span class="author">No name</span>
<span class="author">No name</span>
Line 113: Line 154:
|2cuts||16&micro;L||0&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||14&micro;L||30&micro;L
|2cuts||16&micro;L||0&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||14&micro;L||30&micro;L
|-
|-
-
|NC||0.8&micro;L||0&micro;L||||0&micro;L||0&micro;L||1&micro;L||1&micro;L||7.2&micro;L||10&micro;L
+
|NC||0.8&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||7.2&micro;L||10&micro;L
|}
|}
Line 124: Line 165:
|-
|-
|}
|}
 +
[[File:igku_0922_E7.jpg]]<br>
===Gel Extraction===
===Gel Extraction===
Line 131: Line 173:
!Lane||DNA||Enzyme
!Lane||DNA||Enzyme
|-
|-
-
|1||1kbp ladder||--
+
|1||100bp ladder||--
|-
|-
-
|3~5||Pcon-PT181 antisense (EcoRI&SpaI)||--
+
|2~4||Pcon-attenuator PT181(E-0)||--
 +
|}
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1||100bp ladder||--
 +
|-
 +
|2~4||Pcon-attenuator PT181(E-0)||--
 +
|}
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|2||100bp ladder||--
 +
|-
 +
|4~6||Pcon-attenuator PT181(2-SA)||--
 +
|}
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1||100bp ladder||--
 +
|-
 +
|3~5||spinach-DT(3-2)||--
 +
|-
 +
|8~10||Ptet-antisense PT181(1-3)||--
|}
|}
</div>
</div>
 +
 +
{| class="wikitable"
 +
!Name||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|Pcon-attenuator PT181(E-0)||2.4||1.80||0.17
 +
|-
 +
|Pcon-attenuator PT181(2-SA)||10.8||1.85||0.54
 +
|-
 +
|spinach-DT(3-2)||36.7||1.80||0.75
 +
|-
 +
|Ptet-antisense PT181(1-3)||16.8||1.74||0.78
 +
|}
 +
[[File:Igku 0922 G5.jpg]]<br>
 +
[[File:Igku 0922 G6.jpg]]<br>
 +
[[File:Igku 0922 G7.jpg]]<br>
 +
[[File:Igku 0922 G8.jpg]]<br>
 +
[[File:Igku 0922 G9.jpg]]<br>
 +
[[File:Igku 0922 G10.jpg]]<br>
===Colony PCR===
===Colony PCR===
Line 154: Line 237:
|PconPT181antisense-spinach-DT(9~15)||723
|PconPT181antisense-spinach-DT(9~15)||723
|}
|}
-
 
+
[[File:igku_0922_E8.jpg]]<br>
===Ligation===
===Ligation===
Line 162: Line 245:
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
|-
|-
-
|experiment||9/13 Pcon-PT181 attenuator (SpeI & PstI)||1.7 &micro;L||9/18 aptamer-1R-DT (XbaI & PstI)||5.4&micro;L|| 7.1&micro;L
+
|experiment||9/13 Pcon-PT181 attenuator (SpeI & PstI)||1.7 &micro;L||9/18 aptamer-1R-DT (XbaI & PstI)||5.4&micro;L|| 3.5&micro;L
|-
|-
-
|experiment||9/12 Plac(SpeI & PstI)||2.4 &micro;L||9/18 aptamer-1R-DT (XbaI & PstI)||6.1&micro;L||8.5 &micro;L
+
|experiment||9/12 Plac(SpeI & PstI)||2.4 &micro;L||9/18 aptamer-1R-DT (XbaI & PstI)||6.1&micro;L||3.5 &micro;L
|-
|-
-
|experiment||9/13 Ptet(SpeI & PstI)||1.7 &micro;L||9/10 Plux-RBS-GFP-DT||7.0 &micro;L||8.7 &micro;L
+
|experiment||9/13 Ptet(SpeI & PstI)||1.7 &micro;L||9/10 Plux-RBS-GFP-DT||7.0 &micro;L||4.4 &micro;L
|-
|-
-
|experiment||9/21 RBS-GFP-DT (EcoRI & XbaI)||0.6 &micro;L||9/18 aptamer 12-1R-DT (XbaI & PstI)||5.6&micro;L||6.2 &micro;L
+
|experiment||9/21 RBS-GFP-DT (EcoRI & XbaI)||0.6 &micro;L||9/18 aptamer 12-1R-DT (XbaI & PstI)||5.6&micro;L||3.1 &micro;L
|-
|-
-
|experiment||9/13 PSB1C3(XbaI & PstI)||1.9 &micro;L||9/16 PT181 attenuator(XbaI & PstI)||4.6&micro;L||6.5 &micro;L
+
|experiment||9/13 PSB1C3(XbaI & PstI)||1.9 &micro;L||9/16 PT181 attenuator(XbaI & PstI)||4.6&micro;L||3.3 &micro;L
|-
|-
|}
|}
Line 206: Line 289:
! ||9/7 Pcon-RBS-tetR-DT||EcoRI||XbaI||SpeI||PstI||BufferD||BSA||MilliQ||total
! ||9/7 Pcon-RBS-tetR-DT||EcoRI||XbaI||SpeI||PstI||BufferD||BSA||MilliQ||total
|-
|-
-
|2cut||14&micro;L||1&micro;L||1&micro;L||0&micro;L||0&micro;L||3&micro;L||18.2&micro;L||30&micro;L
+
|2cut||14&micro;L||1&micro;L||1&micro;L||0&micro;L||0&micro;L||3&micro;L||3&micro;L||18.2&micro;L||30&micro;L
|-
|-
|NC||0.7&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||7.3&micro;L||10&micro;L
|NC||0.7&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||7.3&micro;L||10&micro;L
Line 228: Line 311:
|}
|}
incubate 37&deg;C 1hour</div>
incubate 37&deg;C 1hour</div>
 +
[[File:Igku 0922 E9.jpg]]<br>
===Gel Extraction===
===Gel Extraction===
<div class="experiment">
<div class="experiment">
-
<span class="author">Nakamoto</span>
+
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
!Lane||DNA||Enzyme
!Lane||DNA||Enzyme
|-
|-
-
|1||100bp ladder||--
+
|1||1kbp ladder||--
-
|-
+
-
|3||RBS-lacZ&alpha;-DT||XbaI&PstI
+
-
|-
+
-
|4||RBS-lacZ&alpha;-DT||XbaI&PstI
+
-
|-
+
-
|5||RBS-lacZ&alpha;-DT||XbaI&PstI
+
-
|-
+
-
|6||RBS-lacZ&alpha;-DT||XbaI&PstI
+
-
|}
+
-
 
+
-
[[File:igku_Aug19electrophoresis3]]<br>
+
-
[[File:igku_Aug19electrophoresis4]]<br>
+
-
{| class="wikitable"
+
-
!Name||concentration[&micro;g/mL]||260/280||260/230
+
-
|-
+
-
|RBS-lacZ&alpha;-DT(XbaI&PstI)||9.8||1.50||0.39
+
|-
|-
 +
|3~5||Pcon-PT181 antisense (EcoRI&SpaI)||--
|}
|}
</div>
</div>
-
 
+
[[File:Igku 0922 G11.jpg]]<br>
 +
[[File:Igku 0922 G12.jpg]]<br>
===Transformation===
===Transformation===
<div class="experiment">
<div class="experiment">
-
<span class="author">Tatsui</span>
+
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
!Name||Sample||Competent Cells||Plate
!Name||Sample||Competent Cells||Plate
|-
|-
-
|9/17 pSB1C3-Pcon-pT181 attenuator-DT||2 &micro;L||20 &micro;L||CP
+
|9/20 Plac-aptamer12-1R-DT(XbaI&PstI)||2 &micro;L||20 &micro;L||Amp
|-
|-
-
|9/17 pSB1C3-Pcon-aptamer 12_1R-DT||2 &micro;L||20 &micro;L||CP
+
|9/20 pT181 attenuator(1C3)(EcoRI&SpeI)||2 &micro;L||20 &micro;L||CP
|-
|-
-
|9/17 pSB1C3-Pcon-antisense-spinach-DT||2 &micro;L||20 &micro;L||CP
+
|9/17 pT181 attenuator(1C3)(XbaI&PstI)||2 &micro;L||20 &micro;L||CP
|-
|-
-
|9/17 pSB1C3-Pcon-RBS-tetR-DT x2||2 &micro;L||20 &micro;L||CP
+
|9/20 Pcon-pT181 attenuator(SpaI&PstI)＋aptamer12-1R-DT(XbaI&PstI)||2 &micro;L||20 &micro;L||Amp
|-
|-
-
|9/14 pSB1C3-PkaiBC||2 &micro;L||20 &micro;L||CP
+
|kaiABC||2 &micro;L||20 &micro;L||Amp
|-
|-
-
|9/14 pSB1C3-RpaB||2 &micro;L||20 &micro;L||CP
+
|9/22 pSB1C3-RpaB(XbaI&PstI)||2 &micro;L||20 &micro;L||Amp
|-
|-
-
|9/16 pSB1C3-pT181 attenuator(EcoRI&SpeI) ||2 &micro;L||20 &micro;L||CP
+
|9/22 pSB1C3-pT181 attenuator(XbaI&PstI) ||2 &micro;L||20 &micro;L||Amp
|-
|-
-
|9/17 pSB1C3-pT181 attenuator(XbaI&PstI) ||2 &micro;L||20 &micro;L||CP
+
|9/22 Ptet(SpaI&PstI)＋RBS-GFP-DT(XbaI&PstI)||2 &micro;L||20 &micro;L||CP
|-
|-
-
|9/17 pSB1C3-Pcon-spinach-DT||2 &micro;L||20 &micro;L||CP
+
|9/22 RBS-GFP-DT＋Pcon-attenuator pT181 (EcoRI&SpeI)||2 &micro;L||20 &micro;L||CP
|-
|-
-
|9/14 RBS-GFP-DT-PkaiBC||2 &micro;L||20 &micro;L||CP
+
|9/22 pT181 attenuator(XbaI&PstI)(1C3)||2 &micro;L||20 &micro;L||CP
-
|-
+
-
|Plux-RBS-lysis3-DT||2 &micro;L||20 &micro;L||CP
+
-
|-
+
-
|9/20 Ptet-pT181 antisense-spinach-DT||2 &micro;L||20 &micro;L||CP
+
-
|-
+
-
|9/20 Pcon-spinach-DT(pSB4K5) ||2 &micro;L||20 &micro;L||Kan
+
-
|-
+
-
|9/20 Pcon-pT181 antisense-spinach-DT(pSB4K5) ||2 &micro;L||20 &micro;L||Kan
+
-
|-
+
-
|9/20 KaiABC||2 &micro;L||20 &micro;L||Kan
+
-
|-
+
-
|9/20 KaiABC||2 &micro;L||20 &micro;L||Kan
+
-
|-
+
-
|9/20 Plac-RBS-lysis3-DT||2 &micro;L||20 &micro;L||Amp
+
-
|-
+
-
|9/20 Plac-aptamer 12_1R||2 &micro;L||20 &micro;L||Amp
+
-
|-
+
-
|9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT||2 &micro;L||20 &micro;L||Amp
+
-
|-
+
-
|Plac-aptamer12_1R-DT||2 &micro;L||20 &micro;L||Amp
+
-
|-
+
-
|9/20 Plac-aptamer12_1R-DT||2 &micro;L||20 &micro;L||Amp
+
-
|-
+
-
|9/20Pcon-pT181attenuator-aptamer-DT||2 &micro;L||20 &micro;L||Amp
+
-
|-
+
-
|9/17 Plac-pT181-attenuatorx2||2 &micro;L||20 &micro;L||Amp
+
-
|-
+
-
|9/15 Pcon-RBS-lacZ&alpha;-DT||2 &micro;L||20 &micro;L||Amp
+
-
|-
+
-
|9/14 Pcon-pT181attenuator-RBS-lacZ&alpha;-DT||2 &micro;L||20 &micro;L||Amp
+
-
|-
+
-
|PUC19||2 &micro;L||20 &micro;L||Amp
+
|}
|}
</div>
</div>
Line 319: Line 357:
===PCR===
===PCR===
<div class="experiment">
<div class="experiment">
-
<span class="author">Tatsui</span>
+
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
!Sample||base pair
!Sample||base pair
|-
|-
-
|pT181 attenuator(pSB1C3)||--
+
|Pcon-RBS-tetR-DT(1-2(A))||--
|-
|-
-
|Pcon-RBS-lacZ&alpha;-DT||--
+
|Pcon-RBS-tetR-DT(3-2(A))||--
 +
|-
 +
|Pcon-RBS-GFP-DT(1-S(A))||--
 +
|-
 +
|Pcon-RBS-GFP-DT(2-S(A))||--
 +
|-
 +
|Pcon-tetR-DT(E-2(A))||--
|}
|}
{| class="wikitable"
{| class="wikitable"
!PreDenature||Denature||Annealing||Extension||cycle
!PreDenature||Denature||Annealing||Extension||cycle
|-
|-
-
|94 &deg;C||94 &deg;C||55 &deg;C||68 &deg;C||--
+
|94 &deg;C||94 &deg;C||57 &deg;C||68 &deg;C||--
|-
|-
-
|5 min||30 s||30 s||3 min||30 cycles
+
|2 min||10 s||30 s||1min5s||30 cycles
|}
|}
</div>
</div>
 +
[[File:Igku 0922 E10.jpg]]<br>
-
 
+
===Electrophoresis===
-
===Transformation===
+
<div class="experiment">
<div class="experiment">
-
<span class="author">Tatsui</span>
+
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
-
!Name||Sample||Competent Cells||Plate
+
!Lane||Sample
|-
|-
-
|9/17 pSB1C3-Pcon-pT181 attenuator-DT||2 &micro;L||20 &micro;L||CP
+
|1||1kbp ladder
|-
|-
-
|9/17 pSB1C3-Pcon-aptamer 12_1R-DT||2 &micro;L||20 &micro;L||CP
+
|2||Pcon-RBS-tetR-DT(1-2Ⓐ)
|-
|-
-
|9/17 pSB1C3-Pcon-antisense-spinach-DT||2 &micro;L||20 &micro;L||CP
+
|3||Pcon-RBS-tetR-DT(3-2Ⓐ)
|-
|-
-
|9/17 pSB1C3-Pcon-RBS-tetR-DT x2||2 &micro;L||20 &micro;L||CP
+
|4||Pcon-RBS-GFP-DT(1-SⒶ)
|-
|-
-
|9/14 pSB1C3-PkaiBC||2 &micro;L||20 &micro;L||CP
+
|5||Pcon-RBS-GFP-DT(2-SⒶ)
|-
|-
-
|9/14 pSB1C3-RpaB||2 &micro;L||20 &micro;L||CP
+
|6||Pcon-tetR-DT(E-2Ⓐ)
-
|-
+
-
|9/16 pSB1C3-pT181 attenuator(EcoRI&SpeI) ||2 &micro;L||20 &micro;L||CP
+
-
|-
+
-
|9/17 pSB1C3-pT181 attenuator(XbaI&PstI) ||2 &micro;L||20 &micro;L||CP
+
-
|-
+
-
|9/17 pSB1C3-Pcon-spinach-DT||2 &micro;L||20 &micro;L||CP
+
-
|-
+
-
|9/14 RBS-GFP-DT-PkaiBC||2 &micro;L||20 &micro;L||CP
+
-
|-
+
-
|Plux-RBS-lysis3-DT||2 &micro;L||20 &micro;L||CP
+
-
|-
+
-
|9/20 Ptet-pT181 antisense-spinach-DT||2 &micro;L||20 &micro;L||CP
+
-
|-
+
-
|9/20 Pcon-spinach-DT(pSB4K5) ||2 &micro;L||20 &micro;L||Kan
+
-
|-
+
-
|9/20 Pcon-pT181 antisense-spinach-DT(pSB4K5) ||2 &micro;L||20 &micro;L||Kan
+
-
|-
+
-
|9/20 KaiABC||2 &micro;L||20 &micro;L||Kan
+
-
|-
+
-
|9/20 KaiABC||2 &micro;L||20 &micro;L||Kan
+
-
|-
+
-
|9/20 Plac-RBS-lysis3-DT||2 &micro;L||20 &micro;L||Amp
+
-
|-
+
-
|9/20 Plac-aptamer 12_1R||2 &micro;L||20 &micro;L||Amp
+
-
|-
+
-
|9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT||2 &micro;L||20 &micro;L||Amp
+
-
|-
+
-
|Plac-aptamer12_1R-DT||2 &micro;L||20 &micro;L||Amp
+
-
|-
+
-
|9/20 Plac-aptamer12_1R-DT||2 &micro;L||20 &micro;L||Amp
+
-
|-
+
-
|9/20Pcon-pT181attenuator-aptamer-DT||2 &micro;L||20 &micro;L||Amp
+
-
|-
+
-
|9/17 Plac-pT181-attenuatorx2||2 &micro;L||20 &micro;L||Amp
+
-
|-
+
-
|9/15 Pcon-RBS-lacZ&alpha;-DT||2 &micro;L||20 &micro;L||Amp
+
-
|-
+
-
|9/14 Pcon-pT181attenuator-RBS-lacZ&alpha;-DT||2 &micro;L||20 &micro;L||Amp
+
-
|-
+
-
|PUC19||2 &micro;L||20 &micro;L||Amp
+
|}
|}
-
</div>
+
[[File:Igku 0922 E10.jpg]]<br>

Latest revision as of 07:02, 27 September 2013

Contents

Sep 22

Colony PCR

Tatsui

Samplebase pair
9/20 Plac-PT181 attenuator(1~4)664
9/20 Plac-PT181 antisense(1~6)369
9/20 Pcon-PT181 attenuator-aptamer-DT(1~6)859
9/20 Pcon-PT181 antisense-spinach-DT(1~8)723

Igku 0903 E1.jpg
Igku 0922 E2.jpg

Electrophoresis

Nakamoto

LaneSample
1Pcon-apt12_1R-DT(E-1A)
2Pcon-spinach-DT(E-1A)
3Pcon-pT181 attenuator(E-1A)
4DT(1-3)
5tetR aptamer 12_1R-DT(1-3)
6100bp ladder
7Pcon-pT181 attenuator(1-SA)
8spinach-DT(2-1)
9Pcon-pT181 attenuator(E-0)
10Pcon-pT181 attenuator(2-SA)
11spinach-DT(3-2)
12Ptet-PT181 anisense

Igku 0922 E3.jpg
Igku 0922 E4.jpg

Miniprep

Honda

DNAconcentration[µg/mL]260/280260/230
9/21 spinach-DT197.91.671.54
9/21 Plux125.91.711.27
9/21 Plux-RBS-GFP-DT-(1)168.01.621.73
9/21 Pcon-PT181 attenuator-(1)368.31.571.62
9/21 RBS-GFP-DT532.81.751.78
9/21 Pcon-RBS-GFP-DT845.22.021.70

Colony PCR

Honda

Samplebase pair
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1~2)283
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s2min10s30cycles

Igku 0922 E5.jpg
Igku 0922 E6.jpg

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
2~4Pcon-aptamer12-1R-DT(E-1A)--
6~8Pcon-spinach-DT(E-1A)--
10~12Pcon-attenuator PT181(E-1A)--
LaneDNAEnzyme
1100bp ladder--
2~4DT(1-3)--
6~8aptamer12-1RDT(1-3)--
10~12Pcon-PT181 attenuator(1-5A)--
Nameconcentration[µg/mL]260/280260/230
Pcon-aptamer12-1R-DT(E-1A)31.21.870.30
Pcon-spinach-DT(E-1A)25.61.880.99
Pcon-attenuator PT181(E-1A)25.31.990.86
DT(1-3)22.11.820.20
aptamer12-1RDT(1-3)33.51.990.05
Pcon-PT181 attenuator(1-5A)36.91.120.96

Igku 0922 G1.jpg
Igku 0922 G2.jpg
Igku 0922 G3.jpg
Igku 0922 G4.jpg

Restriction Enzyme Digestion

No name

9/16 PlacXbaISpeIPstIBufferBBSAMilliQtotal
2cuts12.5µL0µL1µL1µL3µL3µL9.5µL30µL
NC0.8µL0µL0µL0µL1µL1µL7.4µL10µL
9/22 PluxXbaISpeIPstIBufferBBSAMilliQtotal
2cuts16µL0µL1µL1µL3µL3µL14µL30µL
NC0.8µL0µL0µL0µL1µL1µL7.2µL10µL
9/20 RBS-tetR-2R-DT XbaISpeIPstIBufferDBSAMilliQtotal
2cuts8.5µL1µL0µL1µL3µL3µL13.5µL30µL
NC0.4µL0µL0µL0µL1µL1µL7.6µL10µL

Igku 0922 E7.jpg

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
2~4Pcon-attenuator PT181(E-0)--
LaneDNAEnzyme
1100bp ladder--
2~4Pcon-attenuator PT181(E-0)--
LaneDNAEnzyme
2100bp ladder--
4~6Pcon-attenuator PT181(2-SA)--
LaneDNAEnzyme
1100bp ladder--
3~5spinach-DT(3-2)--
8~10Ptet-antisense PT181(1-3)--
Nameconcentration[µg/mL]260/280260/230
Pcon-attenuator PT181(E-0)2.41.800.17
Pcon-attenuator PT181(2-SA)10.81.850.54
spinach-DT(3-2)36.71.800.75
Ptet-antisense PT181(1-3)16.81.740.78

Igku 0922 G5.jpg
Igku 0922 G6.jpg
Igku 0922 G7.jpg
Igku 0922 G8.jpg
Igku 0922 G9.jpg
Igku 0922 G10.jpg

Colony PCR

No name

Samplebase pair
9/20 Pcon-PT181 attenuator-aptamer12-1R-DT-(7~14)859

Colony PCR

Tatsui

Samplebase pair
PconPT181antisense-spinach-DT(9~15)723

Igku 0922 E8.jpg

Ligation

No name

stateVectorInserterLigation High ver.2
experiment9/13 Pcon-PT181 attenuator (SpeI & PstI)1.7 µL9/18 aptamer-1R-DT (XbaI & PstI)5.4µL 3.5µL
experiment9/12 Plac(SpeI & PstI)2.4 µL9/18 aptamer-1R-DT (XbaI & PstI)6.1µL3.5 µL
experiment9/13 Ptet(SpeI & PstI)1.7 µL9/10 Plux-RBS-GFP-DT7.0 µL4.4 µL
experiment9/21 RBS-GFP-DT (EcoRI & XbaI)0.6 µL9/18 aptamer 12-1R-DT (XbaI & PstI)5.6µL3.1 µL
experiment9/13 PSB1C3(XbaI & PstI)1.9 µL9/16 PT181 attenuator(XbaI & PstI)4.6µL3.3 µL

Liquid Culture

No name

Samplemedium
9/20 Plac-antisense-1--
9/20 Plac-attenuator-1--
9/20 Plac-antisense-1--
9/20 Plac-attenuator-1--

digestion

No name

9/8 Pcon-PT181 antisenseEcoRISpeI BufferMMilliQtotal
2cuts8.3µL1µL1µL3µL16.7µL30µL
NC0.4µL0µL0µL1µL8.3µL10µL
9/7 Pcon-RBS-tetR-DTEcoRIXbaISpeIPstIBufferDBSAMilliQtotal
2cut14µL1µL1µL0µL0µL3µL3µL18.2µL30µL
NC0.7µL0µL0µL0µL0µL1µL1µL7.3µL10µL
9/22 RBS-GFP-DTEcoRIXbaISpeIPstI BufferDBSAMilliQtotal
2cuts3.8µL1µL1µL0µL0µL3µL3µL18.2µL30µL
NC0.2µL0µL0µL0µL0µL1µL1µL7.8µL10µL
9/6 aptamer12-1R-DTEcoRIXbaISpeIPstIBufferDBSAMilliQ
2cut8µL1µL1µL0µL0µL3µL3µL16µL
NC0.4µL0µL0µL0µL0µL1µL1µL7.6µL
incubate 37°C 1hour

Igku 0922 E9.jpg

Gel Extraction

No name

LaneDNAEnzyme
11kbp ladder--
3~5Pcon-PT181 antisense (EcoRI&SpaI)--

Igku 0922 G11.jpg
Igku 0922 G12.jpg

Transformation

No name

NameSampleCompetent CellsPlate
9/20 Plac-aptamer12-1R-DT(XbaI&PstI)2 µL20 µLAmp
9/20 pT181 attenuator(1C3)(EcoRI&SpeI)2 µL20 µLCP
9/17 pT181 attenuator(1C3)(XbaI&PstI)2 µL20 µLCP
9/20 Pcon-pT181 attenuator(SpaI&PstI)＋aptamer12-1R-DT(XbaI&PstI)2 µL20 µLAmp
kaiABC2 µL20 µLAmp
9/22 pSB1C3-RpaB(XbaI&PstI)2 µL20 µLAmp
9/22 pSB1C3-pT181 attenuator(XbaI&PstI) 2 µL20 µLAmp
9/22 Ptet(SpaI&PstI)＋RBS-GFP-DT(XbaI&PstI)2 µL20 µLCP
9/22 RBS-GFP-DT＋Pcon-attenuator pT181 (EcoRI&SpeI)2 µL20 µLCP
9/22 pT181 attenuator(XbaI&PstI)(1C3)2 µL20 µLCP

PCR

No name

Samplebase pair
Pcon-RBS-tetR-DT(1-2(A))--
Pcon-RBS-tetR-DT(3-2(A))--
Pcon-RBS-GFP-DT(1-S(A))--
Pcon-RBS-GFP-DT(2-S(A))--
Pcon-tetR-DT(E-2(A))--
PreDenatureDenatureAnnealingExtensioncycle
94 °C94 °C57 °C68 °C--
2 min10 s30 s1min5s30 cycles

Igku 0922 E10.jpg

Electrophoresis

No name

LaneSample
11kbp ladder
2Pcon-RBS-tetR-DT(1-2Ⓐ)
3Pcon-RBS-tetR-DT(3-2Ⓐ)
4Pcon-RBS-GFP-DT(1-SⒶ)
5Pcon-RBS-GFP-DT(2-SⒶ)
6Pcon-tetR-DT(E-2Ⓐ)
Igku 0922 E10.jpg
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