Template:Kyoto/Notebook/Sep 4

From 2013.igem.org

(Difference between revisions)
(→Transformation)
(→Electrophoresis)
 
(44 intermediate revisions not shown)
Line 12: Line 12:
|experiment||9/3 DT(EcoRI&XbaI) ||3.0µL ||9/3 RBS-lysis3||8.8µL ||4.0µL
|experiment||9/3 DT(EcoRI&XbaI) ||3.0µL ||9/3 RBS-lysis3||8.8µL ||4.0µL
|-
|-
-
|experiment||9/3 DT(EcoRI&XbaI) ||3.0µL ||9/3 aptamer12-1R(EcoRI&SpeI) ||1.9µL ||2.45µL
+
|experiment||9/3 DT(EcoRI&XbaI) ||3.0µL ||9/3 aptamer12_1R(EcoRI&SpeI) ||1.9µL ||2.45µL
|-
|-
|experiment||9/2 pSB1C3(XbaI&PstI)||10.6µL ||9/3 pT181attenuator (XbaI& PstI)||3.2µL ||4.0µL
|experiment||9/2 pSB1C3(XbaI&PstI)||10.6µL ||9/3 pT181attenuator (XbaI& PstI)||3.2µL ||4.0µL
Line 18: Line 18:
|experiment||9/2 pSB1C3(XbaI&PstI)||9.6µL ||9/3 pT181antisense (XbaI& PstI)||2.1µL ||4.0µL
|experiment||9/2 pSB1C3(XbaI&PstI)||9.6µL ||9/3 pT181antisense (XbaI& PstI)||2.1µL ||4.0µL
|-
|-
-
|experiment||9/2 pSB1C3(EcoRI&SpeI)||16.7µL ||9/3 aptamer12-1R(EcoRI&SpeI) ||2.0µL ||4.0µL
+
|experiment||9/2 pSB1C3(EcoRI&SpeI)||16.7µL ||9/3 aptamer12_1R(EcoRI&SpeI) ||2.0µL ||4.0µL
|}
|}
incubate 16 °C 1 hour  
incubate 16 °C 1 hour  
</div>
</div>
-
 
-
 
-
 
-
 
-
 
-
 
-
 
===Transformation===
===Transformation===
<div class="experiment">
<div class="experiment">
Line 41: Line 34:
|9/4 RBS-lysis3-DT ||2&micro;L||20&micro;L||Amp
|9/4 RBS-lysis3-DT ||2&micro;L||20&micro;L||Amp
|-
|-
-
|9/4 aptamer12-1R-DT||2&micro;L||20&micro;L||Amp
+
|9/4 aptamer12_1R-DT||2&micro;L||20&micro;L||Amp
|-
|-
|9/4 pT181attenuator –pSB1C3||2&micro;L||20&micro;L||Amp
|9/4 pT181attenuator –pSB1C3||2&micro;L||20&micro;L||Amp
Line 47: Line 40:
|9/4 pT181antisense-pSB1C3 ||2&micro;L||20&micro;L||Amp
|9/4 pT181antisense-pSB1C3 ||2&micro;L||20&micro;L||Amp
|-
|-
-
|9/4 aptamer12-1R –pSB1C3||2&micro;L||20&micro;L||Amp
+
|9/4 aptamer12_1R –pSB1C3||2&micro;L||20&micro;L||Amp
|-
|-
|9/4 pSB4K5(2013plate5 56) ||2&micro;L||20&micro;L||Amp
|9/4 pSB4K5(2013plate5 56) ||2&micro;L||20&micro;L||Amp
Line 83: Line 76:
|3||8/10 Plac(CP)-2
|3||8/10 Plac(CP)-2
|}  
|}  
-
</div>
 
-
===Master Plate===
 
-
<!-- ここから -->
 
-
<div class="experiment">
 
-
<span class="author">Hirano</span>
 
{| class="wikitable"
{| class="wikitable"
!Number||Use LB plate (+Amp)
!Number||Use LB plate (+Amp)
Line 98: Line 86:
|}  
|}  
</div>
</div>
-
 
===Liquid Culture===
===Liquid Culture===
Line 113: Line 100:
===PCR===
===PCR===
-
<!-- ここから -->
 
<div class="experiment">
<div class="experiment">
<span class="author">Hirano</span>
<span class="author">Hirano</span>
{| class="wikitable"
{| class="wikitable"
-
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||F primer (RBS-luxI-DT-cloning-fond) || R primer(RBS-luxI-DT-cloning-rex)||MilliQ||total
+
!8/29 P&lambda;-RBS-luxI-DT||KOD plus||10x buffer||dNTP||MgSO4||F primer (RBS-luxI-DT-cloning-fwd) || R primer(RBS-luxI-DT-cloning-rev)||MilliQ||total
|-
|-
-
|8/29 p&lambda;-RBS-luxI-DT||0.3&micro;L||0.5&micro;L ||2.5&micro;L ||2.5&micro;L ||1.5&micro;L ||0.75&micro;L ||0.75&micro;L ||16.2&micro;L ||25&micro;L
+
|0.3&micro;L||0.5&micro;L ||2.5&micro;L ||2.5&micro;L ||1.5&micro;L ||0.75&micro;L ||0.75&micro;L ||16.2&micro;L ||25&micro;L
|}
|}
{| class="wikitable"
{| class="wikitable"
-
!PreDenature||Denature||Annealing||Extension||cycle
+
!8/31 Pbad/araC-RBS-RFP(1)||KOD plus||10x buffer||dNTP||MgSO4||F primer (Pbad/araC-pSB1C3-cloning-fond) ||R primer(Pbad/araC-cloning-rex)||MilliQ||total
-
|-
+
-
|　94&deg;C||　98&deg;C||　65&deg;C||　68&deg;C||--
+
-
|-
+
-
|5min||10s　||30s　||3min10s　||30cycles
+
-
|}
+
-
</div>
+
-
<!-- ここまでをコピーしてね -->
+
-
 
+
-
===PCR===
+
-
<!-- ここから -->
+
-
<div class="experiment">
+
-
<span class="author">Hirano</span>
+
-
{| class="wikitable"
+
-
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||F primer (Pbad/araC-pSB1C3-cloning-fond) ||R primer(Pbad/araC-cloning-rex)||MilliQ||total
+
|-
|-
-
|8/31 Pbad/araC-RBS-RFP(1)||0.3&micro;L||0.5&micro;L ||2.5&micro;L ||2.5&micro;L ||1.5&micro;L ||0.75&micro;L ||0.75&micro;L ||16.2&micro;L ||25&micro;L
+
|0.3&micro;L||0.5&micro;L ||2.5&micro;L ||2.5&micro;L ||1.5&micro;L ||0.75&micro;L ||0.75&micro;L ||16.2&micro;L ||25&micro;L
|}
|}
{| class="wikitable"
{| class="wikitable"
!PreDenature||Denature||Annealing||Extension||cycle
!PreDenature||Denature||Annealing||Extension||cycle
|-
|-
-
|　94&deg;C||　98&deg;C||　65&deg;C||　68&deg;C||--
+
|94&deg;C||98&deg;C||65&deg;C||68&deg;C||--
|-
|-
-
|5min||10s　||30s　||3min10s　||30cycles
+
|5min||10s||30s||3min10s||30cycles
|}
|}
</div>
</div>
-
<!-- ここまでをコピーしてね -->
 
===Restriction Enzyme Digestion===
===Restriction Enzyme Digestion===
-
<!-- こっから -->
 
<div class="experiment">
<div class="experiment">
<span class="author">No name</span>
<span class="author">No name</span>
Line 157: Line 127:
! ||8/21 Pconst||SpeI||PstI||Buffer||BSA||MilliQ||total
! ||8/21 Pconst||SpeI||PstI||Buffer||BSA||MilliQ||total
|-
|-
-
|2 cuts||10.5&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||0.3&micro;L||
+
|2 cuts||10.5&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||0.3&micro;L||14.2&micro;L||30 &micro;L
-
14.2&micro;L||30 &micro;L
+
|-
|-
-
|NC||0.5&mic6/8ro;L||0&micro;L||0&micro;||1.0&micro;L||0.1&micro;L|| 8.4&micro;L||10 &micro;L
+
|NC||0.5&micro;L||0&micro;L||0&micro;||1.0&micro;L||0.1&micro;L||8.4&micro;L||10 &micro;L
|}
|}
Line 166: Line 135:
! ||8/29 Plac||SpeI||PstI||Buffer||BSA||MilliQ||total
! ||8/29 Plac||SpeI||PstI||Buffer||BSA||MilliQ||total
|-
|-
-
|2 cuts||13.0&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||0.3&micro;L||
+
|2 cuts||13.0&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||0.3&micro;L||11.7&micro;L||30 &micro;L
-
11.7&micro;L||30 &micro;L
+
|-
|-
-
|NC||0.7&mic6/8ro;L||0&micro;L||0&micro;||1.0&micro;L||0.1&micro;L|| 8.2&micro;L||10 &micro;L
+
|NC||0.7&micro;L||0&micro;L||0&micro;||1.0&micro;L||0.1&micro;L|| 8.2&micro;L||10 &micro;L
|}
|}
Line 175: Line 143:
! ||8/20 Ptet||SpeI||PstI||Buffer||BSA||MilliQ||total
! ||8/20 Ptet||SpeI||PstI||Buffer||BSA||MilliQ||total
|-
|-
-
|2 cuts||9.0&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||0.3&micro;L||
+
|2 cuts||9.0&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||0.3&micro;L||15.7&micro;L||30 &micro;L
-
15.7&micro;L||30 &micro;L
+
|-
|-
-
|NC||0.5&mic6/8ro;L||0&micro;L||0&micro;||1.0&micro;L||0.1&micro;L|| 8.4&micro;L||10 &micro;L
+
|NC||0.5&micro;L||0&micro;L||0&micro;||1.0&micro;L||0.1&micro;L|| 8.4&micro;L||10 &micro;L
|}
|}
Line 184: Line 151:
! ||8/20 RBS-tetR-DT||XbaI||PstI||Buffer||BSA||MilliQ||total
! ||8/20 RBS-tetR-DT||XbaI||PstI||Buffer||BSA||MilliQ||total
|-
|-
-
|2 cuts||8.5&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||0.3&micro;L||
+
|2 cuts||8.5&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||0.3&micro;L||16.2&micro;L||30 &micro;L
-
16.2&micro;L||30 &micro;L
+
|-
|-
-
|NC||0.5&mic6/8ro;L||0&micro;L||0&micro;||1.0&micro;L||0.1&micro;L|| 8.4&micro;L||10 &micro;L
+
|NC||0.5&micro;L||0&micro;L||0&micro;||1.0&micro;L||0.1&micro;L|| 8.4&micro;L||10 &micro;L
|}
|}
Line 193: Line 159:
! ||spinach -DT||XbaI||PstI||Buffer||BSA||MilliQ||total
! ||spinach -DT||XbaI||PstI||Buffer||BSA||MilliQ||total
|-
|-
-
|2 cuts||5.4&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||3.0&micro;L||
+
|2 cuts||5.4&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||3.0&micro;L||16.6&micro;L||30 &micro;L
-
16.6&micro;L||30 &micro;L
+
|-
|-
-
|NC||0.3&mic6/8ro;L||0&micro;L||0&micro;||1.0&micro;L||1.0&micro;L|| 7.7&micro;L||10 &micro;L
+
|NC||0.3&micro;L||0&micro;L||0&micro;||1.0&micro;L||1.0&micro;L|| 7.7&micro;L||10 &micro;L
|}
|}
Line 202: Line 167:
! ||8/17 RBS-lacZ&alpha;-DT ||XbaI||PstI||Buffer||BSA||MilliQ||total
! ||8/17 RBS-lacZ&alpha;-DT ||XbaI||PstI||Buffer||BSA||MilliQ||total
|-
|-
-
|2 cuts||11&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||3.0&micro;L||
+
|2 cuts||11&micro;L||1.0&micro;L||1.0&micro;L||3.0&micro;L||3.0&micro;L||11&micro;L||30 &micro;L
-
11&micro;L||30 &micro;L
+
|-
|-
-
|NC||0.5&mic6/8ro;L||0&micro;L||0&micro;||1.0&micro;L||1.0&micro;L|| 7.5&micro;L||10 &micro;L
+
|NC||0.5&micro;L||0&micro;L||0&micro;||1.0&micro;L||1.0&micro;L||7.5&micro;L||10 &micro;L
|}
|}
</div>
</div>
Line 219: Line 183:
|Ptrc KaiB||1&micro;L||10&micro;L||11&micro;L||Amp
|Ptrc KaiB||1&micro;L||10&micro;L||11&micro;L||Amp
|-
|-
-
|pS&omega; ||1&micro;L||10&micro;L||11&micro;L||Amp
+
|pS&Omega; ||1&micro;L||10&micro;L||11&micro;L||Amp
|}
|}
</div>
</div>
Line 231: Line 195:
!Lane||Sample||Enzyme1||Enzyme2
!Lane||Sample||Enzyme1||Enzyme2
|-
|-
-
|1||100bp ladder||-||--
+
|1||100bp ladder||--||--
|-
|-
|2||Pconst||SpeI||PstI
|2||Pconst||SpeI||PstI
Line 243: Line 207:
|6||100bp ladder||--||--
|6||100bp ladder||--||--
|}
|}
-
[[File:igku_xxxxxx.xxx]]<br>
+
 
2
2
{| class="wikitable"
{| class="wikitable"
!Lane||Sample||Enzyme1||Enzyme2
!Lane||Sample||Enzyme1||Enzyme2
|-
|-
-
|1||100bp ladder||-||--
+
|1||100bp ladder||--||--
|-
|-
|2||Ptet||SpeI||PstI
|2||Ptet||SpeI||PstI
Line 260: Line 224:
|6||100bp ladder||--||--
|6||100bp ladder||--||--
|}
|}
-
[[File:igku_xxxxxx.xxx]]<br>
+
 
 +
3
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme1||Enzyme2
 +
|-
 +
|1||100bp ladder||--||--
 +
|-
 +
|2||Spinach-DT||XbaI||PstI
 +
|-
 +
|3||Spinach-DT NC||--||--
 +
|-
 +
|4||RBS-lacZ&alpha;-DT||XbaI||PstI
 +
|-
 +
|5||RBS-lac&alpha;-DT NC||--||--
 +
|-
 +
|6||100bp ladder||--||--
 +
|}
 +
[[File:Igku_Sep4_Electrophoresis(N1).jpg]]<br>
 +
[[File:Igku_Sep4_Electrophoresis_N2_3.jpg]]<br>
 +
 
</div>
</div>
Line 266: Line 249:
<div class="experiment">
<div class="experiment">
-
<span class="author">No name</span></div>
+
<span class="author">No name</span>
1
1
Line 284: Line 267:
|-
|-
|6||Plac||SpeI&PstI
|6||Plac||SpeI&PstI
-
|-
 
-
 
-
 
-
 
-
|}
 
-
[[File:igku_xxbeforexx.xxx]]<br>
 
-
[[File:igku_xxafterxx.xxx]]<br>
 
-
{| class="wikitable"
 
-
!Name||quantity||concentration[&micro;g/mL]||260/280||260/230
 
-
|-
 
-
|Pcon||--||13.2 ||1.99 ||-1.64
 
-
|-
 
-
|Plac||-- ||15.3 ||2.07 ||-o.39
 
|}
|}
-
 
-
<div class="experiment">
 
-
<span class="author">No name</span></div>
 
2
2
-
 
{| class="wikitable"
{| class="wikitable"
!Lane||DNA||Enzyme
!Lane||DNA||Enzyme
Line 318: Line 284:
|-
|-
|6||RBS-tetR-DT||XbaI&PstI
|6||RBS-tetR-DT||XbaI&PstI
-
|-
 
-
 
-
 
-
 
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:Igku Sep4 Electrophoresis(N2).jpg]]<br>
-
[[File:igku_xxafterxx.xxx]]<br>
+
[[File:Igku Sep4 Gel Extraction(N2) 2.jpg]]<br>
{| class="wikitable"
{| class="wikitable"
!Name||quantity||concentration[&micro;g/mL]||260/280||260/230
!Name||quantity||concentration[&micro;g/mL]||260/280||260/230
|-
|-
-
|Ptet||-- ||30.1 ||1.97 ||-0.97
+
|Pcon||--||13.2 ||1.99 ||-1.64
 +
|-
 +
|Plac||-- ||15.3 ||2.07 ||-0.39
|-
|-
-
|RBS-tetR-DT||-- ||3.0 ||2.44 ||0.88
+
|Ptet||-- ||30.1||1.97 ||-0.97
 +
|-
 +
|RBS-tetR-DT||--||3.0||2.44 ||0.88
|}
|}
 +
</div>
===Gel Extraction===
===Gel Extraction===
-
 
<div class="experiment">
<div class="experiment">
<span class="author">No name</span></div>
<span class="author">No name</span></div>
-
 
-
 
{| class="wikitable"
{| class="wikitable"
!Lane||DNA||Enzyme
!Lane||DNA||Enzyme
Line 344: Line 308:
|1||100bp ladder||--
|1||100bp ladder||--
|-
|-
-
|2||spinach-DT||XbaII&PstI
+
|2||spinach-DT||XbaI&PstI
|-
|-
-
|3||spinach-DT NC||XbaII&PstI
+
|3||spinach-DT ||XbaI&PstI
|-
|-
-
|4||RBS-lacZ&alpha;-DT||XbaI&PstI
+
|4||--||--
|-
|-
-
|5||RBS-lacZ&alpha;-DT NC||XbaI&PstI
+
|5||RBS-lacZ&alpha;-DT||XbaI&PstI
|-
|-
-
|6||100bp ladder ||--
+
|6||RBS-lacZ&alpha;-DT||XbaI&PstI
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:Igku Sep4 Gel Extraction(N3) 1.jpg]]<br>
-
[[File:igku_xxafterxx.xxx]]<br>
+
[[File:Igku Sep4 Gel Extraction(N3) 2.jpg]]<br>
{| class="wikitable"
{| class="wikitable"
!Name||quantity||concentration[&micro;g/mL]||260/280||260/230
!Name||quantity||concentration[&micro;g/mL]||260/280||260/230
|-
|-
-
|spinach-DT||--||2.1 ||2.10 ||-0.05
+
|spinach-DT||--||2.1||2.10||-0.05
|-
|-
-
|RBS-lacZ&alpha;-DT||-- ||3.3 ||2.59 ||-0.07
+
|RBS-lacZ&alpha;-DT||--||3.3||2.59||-0.07
|}
|}
</div>
</div>
Line 378: Line 342:
|-
|-
|}  
|}  
-
 
-
</div>
 
-
 
-
===Master Plate===
 
-
<div class="experiment">
 
-
<span class="author">Hirano</span>
 
{| class="wikitable"
{| class="wikitable"
!Number||Use LB plate
!Number||Use LB plate
Line 394: Line 352:
|-
|-
|4||JM109-4
|4||JM109-4
-
|-
 
|}  
|}  
-
 
-
 
-
 
</div>
</div>
Line 408: Line 362:
|-
|-
|8/9 BBa_J23100-3||Plusgrow medium(+Amp)
|8/9 BBa_J23100-3||Plusgrow medium(+Amp)
-
|-
 
-
|}
 
-
</div>
 
-
 
-
===Liquid Culture===
 
-
<div class="experiment">
 
-
<span class="author">Hirano</span>
 
-
{| class="wikitable"
 
-
!Sample||medium
 
|-
|-
|JM109-1(Master plate)||LB medium
|JM109-1(Master plate)||LB medium
-
|-
 
|}
|}
</div>
</div>
Line 442: Line 386:
|-
|-
|5min||30s||30s||30s||30cycles
|5min||30s||30s||30s||30cycles
-
|}
 
-
</div>
 
-
 
-
===Gel Extraction===
 
-
 
-
<div class="experiment">
 
-
<span class="author">No name</span></div>
 
-
 
-
 
-
{| class="wikitable"
 
-
!Lane||DNA||Enzyme
 
-
|-
 
-
|1||100bp ladder||--
 
-
|-
 
-
|2||spinach-DT||XbaII&PstI
 
-
|-
 
-
|3||spinach-DT ||XbaII&PstI
 
-
|-
 
-
|4||--||--
 
-
|-
 
-
|5||RBS-lacZ&alpha;-DT||XbaI&PstI
 
-
|-
 
-
|6||RBS-lacZ&alpha;-DT||XbaI&PstI
 
-
|}
 
-
[[File:igku_xxbeforexx.xxx]]<br>
 
-
[[File:igku_xxafterxx.xxx]]<br>
 
-
{| class="wikitable"
 
-
!Name||quantity||concentration[&micro;g/mL]||260/280||260/230
 
-
|-
 
-
|spinach-DT||--||2.1 ||2.10 ||-0.05
 
-
|-
 
-
|RBS-lacZ&alpha;-DT||-- ||3.3 ||2.59 ||-0.07
 
|}
|}
</div>
</div>

Latest revision as of 00:34, 27 September 2013

Contents

Sep 4

Ligation

No name

stateVectorInserterLigation High ver.2
experiment9/3 DT(EcoRI&XbaI)3.2µL9/3 RBS-lysis12.4µL2.7µL
experiment9/3 DT(EcoRI&XbaI) 3.0µL 9/3 RBS-lysis24.7µL 4.0µL
experiment9/3 DT(EcoRI&XbaI) 3.0µL 9/3 RBS-lysis38.8µL 4.0µL
experiment9/3 DT(EcoRI&XbaI) 3.0µL 9/3 aptamer12_1R(EcoRI&SpeI) 1.9µL 2.45µL
experiment9/2 pSB1C3(XbaI&PstI)10.6µL 9/3 pT181attenuator (XbaI& PstI)3.2µL 4.0µL
experiment9/2 pSB1C3(XbaI&PstI)9.6µL 9/3 pT181antisense (XbaI& PstI)2.1µL 4.0µL
experiment9/2 pSB1C3(EcoRI&SpeI)16.7µL 9/3 aptamer12_1R(EcoRI&SpeI) 2.0µL 4.0µL

incubate 16 °C 1 hour

Transformation

Tatsui

NameSampleCompetent CellsPlate
9/4 RBS-lysis1-DT 2µL20µLAmp
9/4 RBS-lysis2-DT 2µL20µLAmp
9/4 RBS-lysis3-DT 2µL20µLAmp
9/4 aptamer12_1R-DT2µL20µLAmp
9/4 pT181attenuator –pSB1C32µL20µLAmp
9/4 pT181antisense-pSB1C3 2µL20µLAmp
9/4 aptamer12_1R –pSB1C32µL20µLAmp
9/4 pSB4K5(2013plate5 56) 2µL20µLAmp
9/3 Mutagenesis product 1(KaiABC) 2µL20µLAmp
9/3 Mutagenesis product 2(NC) 2µL20µLAmp

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
DT193.11.931.81
Spinach-DT373.31.921.69
RBS-lysis1332.01.951.62

Master Plate

Hirano

NumberUse LB plate (+CP)
18/10 Plac(CP)-2
2 8/10 Plac(CP)-2
38/10 Plac(CP)-2
NumberUse LB plate (+Amp)
18/9 J23100(Amp)-1
28/9 J23100(Amp)-1
38/9 J23100(Amp)-1

Liquid Culture

Hirano

Samplemedium
8/10 Plac(CP)-2Plusgrow medium(+CP)
8/9 J23100(Amp)-1Plusgrow medium(+Amp)

PCR

Hirano

8/29 Pλ-RBS-luxI-DTKOD plus10x bufferdNTPMgSO4F primer (RBS-luxI-DT-cloning-fwd) R primer(RBS-luxI-DT-cloning-rev)MilliQtotal
0.3µL0.5µL 2.5µL 2.5µL 1.5µL 0.75µL 0.75µL 16.2µL 25µL
8/31 Pbad/araC-RBS-RFP(1)KOD plus10x bufferdNTPMgSO4F primer (Pbad/araC-pSB1C3-cloning-fond) R primer(Pbad/araC-cloning-rex)MilliQtotal
0.3µL0.5µL 2.5µL 2.5µL 1.5µL 0.75µL 0.75µL 16.2µL 25µL
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C65°C68°C--
5min10s30s3min10s30cycles

Restriction Enzyme Digestion

No name

8/21 PconstSpeIPstIBufferBSAMilliQtotal
2 cuts10.5µL1.0µL1.0µL3.0µL0.3µL14.2µL30 µL
NC0.5µL0µL0µ1.0µL0.1µL8.4µL10 µL
8/29 PlacSpeIPstIBufferBSAMilliQtotal
2 cuts13.0µL1.0µL1.0µL3.0µL0.3µL11.7µL30 µL
NC0.7µL0µL0µ1.0µL0.1µL 8.2µL10 µL
8/20 PtetSpeIPstIBufferBSAMilliQtotal
2 cuts9.0µL1.0µL1.0µL3.0µL0.3µL15.7µL30 µL
NC0.5µL0µL0µ1.0µL0.1µL 8.4µL10 µL
8/20 RBS-tetR-DTXbaIPstIBufferBSAMilliQtotal
2 cuts8.5µL1.0µL1.0µL3.0µL0.3µL16.2µL30 µL
NC0.5µL0µL0µ1.0µL0.1µL 8.4µL10 µL
spinach -DTXbaIPstIBufferBSAMilliQtotal
2 cuts5.4µL1.0µL1.0µL3.0µL3.0µL16.6µL30 µL
NC0.3µL0µL0µ1.0µL1.0µL 7.7µL10 µL
8/17 RBS-lacZα-DT XbaIPstIBufferBSAMilliQtotal
2 cuts11µL1.0µL1.0µL3.0µL3.0µL11µL30 µL
NC0.5µL0µL0µ1.0µL1.0µL7.5µL10 µL

Transformation

No name

NameSampleCompetent CellsTotalPlate
Ptrc KaiC1µL10µL11µLAmp
Ptrc KaiB1µL10µL11µLAmp
pSΩ 1µL10µL11µLAmp

Electrophoresis

No name
1

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2PconstSpeIPstI
3Pconst NC----
4PlacSpeIPstI
5Plac NC----
6100bp ladder----

2

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2PtetSpeIPstI
3Ptet NC----
4RBS-tetR-DTXbaIPstI
5RBS-tetR-DT NC----
6100bp ladder----

3

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2Spinach-DTXbaIPstI
3Spinach-DT NC----
4RBS-lacZα-DTXbaIPstI
5RBS-lacα-DT NC----
6100bp ladder----

Igku Sep4 Electrophoresis(N1).jpg
Igku Sep4 Electrophoresis N2 3.jpg

Gel Extraction

No name

1

LaneDNAEnzyme
1100bp ladder--
2PconSpeI&PstI
3PconSpeI&PstI
4----
5PlacSpeI&PstI
6PlacSpeI&PstI

2

LaneDNAEnzyme
1100bp ladder--
2PtetSpeI&PstI
3PtetSpeI&PstI
4----
5RBS-tetR-DTXbaI&PstI
6RBS-tetR-DTXbaI&PstI

Igku Sep4 Electrophoresis(N2).jpg
Igku Sep4 Gel Extraction(N2) 2.jpg

Namequantityconcentration[µg/mL]260/280260/230
Pcon--13.2 1.99 -1.64
Plac-- 15.3 2.07 -0.39
Ptet-- 30.11.97 -0.97
RBS-tetR-DT--3.02.44 0.88

Gel Extraction

No name
LaneDNAEnzyme
1100bp ladder--
2spinach-DTXbaI&PstI
3spinach-DT XbaI&PstI
4----
5RBS-lacZα-DTXbaI&PstI
6RBS-lacZα-DTXbaI&PstI

Igku Sep4 Gel Extraction(N3) 1.jpg
Igku Sep4 Gel Extraction(N3) 2.jpg

Namequantityconcentration[µg/mL]260/280260/230
spinach-DT--2.12.10-0.05
RBS-lacZα-DT--3.32.59-0.07

Master Plate

No name

NumberUse LB plate (+Amp)
48/9 BBa_J23100-3
58/9 BBa_J23100-4
68/9 BBa_J23100-5
NumberUse LB plate
1JM109-1
2JM109-2
3JM109-3
4JM109-4

Liquid Culture

Hirano

Samplemedium
8/9 BBa_J23100-3Plusgrow medium(+Amp)
JM109-1(Master plate)LB medium

Colony PCR

Hirano

Samplebase pair
8/9 BBa_J23100 3--
8/9 BBa_J23100 4--
8/9 BBa_J23100 5--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30s30cycles
Retrieved from "http://2013.igem.org/Template:Kyoto/Notebook/Sep_4"