Template:Kyoto/Notebook/Sep 14

From 2013.igem.org

(Difference between revisions)
(→Gel Extraction)
 
(19 intermediate revisions not shown)
Line 2: Line 2:
===Restriction Enzyme Digestion===
===Restriction Enzyme Digestion===
<div class="experiment">
<div class="experiment">
-
<span class="author">No name</span>
+
<span class="author">Hirano</span>
{| class="wikitable"
{| class="wikitable"
! ||9/14 pSB4K5S||EcoRI||XbaI||SpeI||PstI||BufferB||BufferD||BSA||MilliQ||total
! ||9/14 pSB4K5S||EcoRI||XbaI||SpeI||PstI||BufferB||BufferD||BSA||MilliQ||total
Line 42: Line 42:
===Liquid Culture===
===Liquid Culture===
-
<!-- ここから -->
 
<div class="experiment">
<div class="experiment">
-
<span class="author">No name</span>
+
<span class="author">Hirano</span>
{| class="wikitable"
{| class="wikitable"
!Sample||medium
!Sample||medium
|-
|-
-
|8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1)||Plusgrow medium(+Amp)
+
|9/11 Plac(BBa-R0011)-(3)||Plusgrow medium(+Amp)
 +
|}
 +
</div>
 +
 
 +
===Electrophoresis===
 +
 +
<div class="experiment">
 +
<span class="author">Hirano</span>
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme1||Enzyme2
|-
|-
-
|8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(2)||Plusgrow medium(+Amp)
+
|1||1kb ladder||--||--
|-
|-
-
|8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(3)||Plusgrow medium(+Amp)
+
|2||pSB4K5(1)||EcoRI||SpeI
|-
|-
-
|8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(4)||Plusgrow medium(+Amp)
+
|3|| pSB4K5(1)||--||--
|-
|-
-
|8/29 Plux-RBS-GFP-DT-(1)||Plusgrow medium(+CP)
+
|4||Plac(1A2)||PstI||--
|-
|-
-
|8/29 Plux-RBS-GFP-DT-(2)||Plusgrow medium(+CP)
+
|5||Plac(1A2)||--||--
|-
|-
-
|8/29 Pbad/araC-RBS-RFP-DT-(1)||Plusgrow medium(+CP)
+
|6||PKaiBC||EcoRI||SpeI
|-
|-
-
|8/29 Pbad-araC-RBS-RFP-DT-(2)||Plusgrow medium(+CP)
+
|7||PKaiBC||--||--
|-
|-
-
|RBS-lysis3-DT-(1)||Plusgrow medium(+CP)
+
|8||RpaB(1)||XbaI||PstI
 +
|-
 +
|9||RpaB(1)||--||--
 +
|-
 +
|10||RBS-GFP-DT||XbaI||--
 +
|-
 +
|11||RBS-GFP-DT||--||--
 +
|-
 +
|12||1kb ladder||--||--
|}
|}
 +
[[File:igku_0914_E1.jpg]]<br>
</div>
</div>
-
 
-
===PCR===
 
-
<!-- ここから -->
 
-
<div class="experiment">
 
-
<span class="author">No name</span>
 
{| class="wikitable"
{| class="wikitable"
-
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||SasA_fwd primer||SasA_rev primer||MilliQ||total
+
!Lane||Sample||Enzyme
|-
|-
-
| ||0.5||2.5||2.5||1.5||0.75||0.75|| ||25
+
|1||1kb ladder||--
-
|}
+
-
{| class="wikitable"
+
-
!PreDenature||Denature||Annealing||Extension||cycle
+
|-
|-
-
|　&deg;C||　&deg;C||　&deg;C||　&deg;C||--
+
|2||Plac(pSB1A2)||PstI
|-
|-
-
|（◎Д◎）　||　||　||　||
+
|3||Plac(pSB1A2)||--
|}
|}
-
</div>
+
[[File:igku_0914_E2.jpg]]<br>
-
<!-- ここまでをコピーしてね -->
+
-
===Restriction Enzyme Digestion===
+
===Sequence PCR===
<div class="experiment">
<div class="experiment">
<span class="author">No name</span>
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
-
! ||8/22 psB1C3-(2)||EcoRI||SpeI||XbaI||PstI||BufferB||BufferD||BSA||MilliQ||total
+
!genome DNA||Big Dye||5x buffer||temp(400mg)||primer||milliQ
|-
|-
-
|2 cuts||8&micro;L||0.5&micro;L||0.5&micro;L||0&micro;L||0&micro;L||3&micro;L||0&micro;L||0.3&micro;L||17.7&micro;L||30&micro;L
+
|Pcon-RBS-luxR-DT||0.5||1.75||0.9||0.5||6.85
|-
|-
-
|NC||1&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0&micro;L||0.1&micro;L||7.9&micro;L||10&micro;L
+
|Pcon-RBS-luxR-DT||0.5||1.75||0.4||0.5||7.35
|-
|-
-
|2 cuts||8&micro;L||0&micro;L||0&micro;L||0.5&micro;L||0.5&micro;L||0&micro;L||3&micro;L||0.3&micro;L||17.7&micro;L||30&micro;L
+
|Pcon-RBS-tetR-DT||0.5||1.75||3||0.5||5.25
|-
|-
-
|NC||1&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||7.9&micro;L||10&micro;L
+
|Pcon-RBS-tetR-DT||0.5||1.75||3||0.5||5.25
-
|}
+
-
{| class="wikitable"
+
-
! ||8/21 tRMA-spinach(1)||XbaI||SpeI||BufferB||BSA||MilliQ||total
+
|-
|-
-
|2 cuts||4&micro;L||0.5&micro;L||0.5&micro;L||3&micro;L||0.3&micro;L||21.7&micro;L||30&micro;L
+
|Plux||0.5||1.75||2.4||0.5||5.35
|-
|-
-
|NC||0.5&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.4&micro;L||10&micro;L
+
|Plux-RBS-GFP-DT||0.5||1.75||1.9||0.5||5.85
 +
|-
 +
|Plux-RBS-GFP-DT||0.5||0.5||1.9||0.5||5.85
 +
|-
 +
|Pcon-RBS-GFP-DT||0.5||0.5||1.2||0.5||6.55
 +
|-
 +
|Pcon-RBS-GFP-DT||0.5||0.5||1.2||0.5||6.55
 +
|-
 +
|RBS-lysis3-DT||0.5||0.5||1.0||0.5||6.75
 +
|-
 +
|Pcon-antisense-Spinach-DT||0.5||0.5||1.4||0.5||6.35
 +
|-
 +
|Pcon-RBS lacZ&alpha;-DT||0.5||0.5||1.4||0.5||6.35
 +
|-
 +
|Ptet-RBS lacZ&alpha;-DT||0.5||0.5||0.59||0.5||1.85
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||8/21 tetR aptamer12-1R(1)||EcoRI||SpeI||BufferB||BSA||MilliQ||total
+
!PreDenature||Denature||Annealing||Extension||cycle
|-
|-
-
|2 cuts||3&micro;L||0.5&micro;L||0.5&micro;L||3&micro;L||0.3&micro;L||22.7&micro;L||30&micro;L
+
|96&deg;C||96&deg;C||50&deg;C||60&deg;C||--
|-
|-
-
|NC||0.4&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.5&micro;L||10&micro;L
+
|2min||10sec||5sec||4min||30cycle
|}
|}
 +
</div>
 +
 +
===Gel Extraction===
 +
<div class="experiment">
 +
<span class="author">Tatsui</span>
{| class="wikitable"
{| class="wikitable"
-
! ||8/21 PT181 attenuator-(2)||EcoRI||SpeI||XbaI||PstI||BufferB||BufferD||BSA||MilliQ||total
+
!Lane||DNA||Enzyme
|-
|-
-
|2 cuts||4&micro;L||0.5&micro;L||0.5&micro;L||0&micro;L||0&micro;L||3&micro;L||0&micro;L||0.3&micro;L||21.7&micro;L||30&micro;L
+
|1||1kb ladder||--
|-
|-
-
|NC||0.5&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0&micro;L||0.1&micro;L||8.4&micro;L||10&micro;L
+
|2||pSB4K5||EcoRI&SpeI
|-
|-
-
|2 cuts||4&micro;L||0&micro;L||0&micro;L||0.5&micro;L||0.5&micro;L||0&micro;L||3&micro;L||0.3&micro;L||21.7&micro;L||30&micro;L
+
|3||pSB4K5||EcoRI&SpeI
|-
|-
-
|NC||0.5&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.4&micro;L||10&micro;L
+
|4||pSB4K5||EcoRI&SpeI
|}
|}
 +
[[File:igku_0914_G8.jpg]]<br>
 +
[[File:igku_0914_G9.jpg]]<br>
 +
 +
===Restriction Enzyme Digestion===
 +
<div class="experiment">
 +
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
-
! ||8/21 PT181 antisense-(2)||EcoRI||SpeI||XbaI||PstI||BufferB||BufferD||BSA||MilliQ||total
+
! ||9/14 Plac(1A2)||EcoRI||XbaI||SpeI||PstI||BufferB||BSA||MilliQ||total
|-
|-
-
|2 cuts||10&micro;L||0.5&micro;L||0.5&micro;L||0&micro;L||0&micro;L||3&micro;L||0&micro;L||0.3&micro;L||15.7&micro;L||30&micro;L
+
|2cuts||23&micro;L||0&micro;L||0&micro;L||0.3&micro;L||0&micro;L||3&micro;L||3&micro;L||0&micro;L||30&micro;L
 +
|}
 +
{| class="wikitable"
 +
! ||9/14 RBS-GFP-DT||EcoRI||XbaI||SpeI||PstI||BufferH||BSA||MilliQ||total
|-
|-
-
|NC||1&micro;L||0&micro;L||0&;L||0&micro;L||0&micro;L||1&micro;L||0&micro;L||0.1&micro;L||7.9&micro;L||10&micro;L
+
|2 cuts||23&micro;L||1&micro;L||0&micro;L||0&micro;L||0&micro;L||3&micro;L||3&micro;L||0&micro;L||30&micro;L
-
|-
+
-
|2 cuts||10&micro;L||0&micro;L||0&micro;L||0.5&micro;L||0.5&micro;L||0&micro;L||3&micro;L||0.3&micro;L||15.7&micro;L||30&micro;L
+
-
|-
+
-
|NC||1&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||7.9&micro;L||10&micro;L
+
|}
|}
-
*incubated 37&deg;C 1hour
 
-
[[File:Igku Aug29 Restriction Enzyme Digestion(N③).jpg]]<br>
 
</div>
</div>
-
 
===Ligation===  
===Ligation===  
Line 148: Line 174:
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
|-
|-
-
|experiment||8/30 pSB1C3 (EcoRI & SpeI)||6.6||8/30 tRNA-Spinach (EcoRI & SpeI)||12.5||3.5
+
|experiment||9/13 Pcon-PT181attenuator(SpeI&PstI)||1.7||9/13 RBS-lacZα-DT (XbaI & PstI)||12.5||5
|-
|-
-
|experiment||8/30 pSB1C3 (EcoRI & SpeI)||6.6||8/30 pT181 antisense (EcoRI & SpeI)||12.5||3.5
+
|experiment||9/13 pSB2C3 (XbaI & PstI)||1.9||9/14 RpaB (XbaI & PstI)||16.3||10.5
|-
|-
-
|experiment||8/30 pSB1C3 (XbaI & Pst)||12.5||8/30 pT181 attenuator (XbaI & PstI)||25||3.5
+
|experiment||9/13 pSB2C3 (XbaI & PstI)||4.7||9/14 PkaiBC (EcoRI & SpeI)||6.7||5.7
|-
|-
-
|experiment||8/30 pSB1C3 (XbaI & Pst)||12.5||8/30 pT181 antisense (XbaI & PstI)||25||3.5
+
|experiment||9/14 RBS-GFP-DT (PstI&XbaI)||0.7||9/14 PkaiBC (EcoRI & SpeI)||2.6||1.6
|-
|-
-
|experiment||8/20 DT (EcoRI & XbaI)||6.6||8/30 pT181 attenuator(2) (EcoRI & SpeI)||12.5||3.5
+
|experiment||9/14 Plac (SpeI&PstI)||2.4||9/14 RBS-lysis1-DT (XbaI & PstI)||4.9||3.6
|-
|-
-
|experiment||8/20 DT (EcoRI & XbaI)||6.6||8/30 pT181 antisense (EcoRI & SpeI)||12.5||3.5
+
|experiment||9/14 Plac (SpeI&PstI)||2.4||9/14 RBS-lysis2-DT (XbaI & PstI)||7.0||4.7
|-
|-
-
|experiment||8/27 DT (EcoRI & XbaI)||3.0||8/30 tRNA-Spinach (EcoRI & SpeI)||12.5||3.5
+
|experiment||9/14 Plac (SpeI&PstI)||2.4||9/14 RBS-lysis3-DT (XbaI & PstI)||9.2||5.8
-
|-
+
-
|experiment||8/20 DT (EcoRI & XbaI)||6.6||8/30 RBS-lysis2 (EcoRI & SpeI)||12.5||3.5
+
-
|-
+
-
|experiment||8/22 J23100 (SpeI & PstI)||--||RBS-tetR-DT (XbaI & PstI)||--||3.5
+
-
|-
+
-
|experiment||8/22 Plac (SpeI & PstI)||8.0||8/18 RBS-lacZ&alpha;-DT (XbaI & PstI)||10||3.5
+
|}
|}
Samples were evaporeted used evaporator into about 7 µL
Samples were evaporeted used evaporator into about 7 µL
</div>
</div>
-
 
===Colony PCR===
===Colony PCR===
Line 179: Line 198:
!Sample||base pair
!Sample||base pair
|-
|-
-
|8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT(8/20 LB Amp) (1)~(4)||2143
+
|9/13 Plux＋RBS-lysis1-DT (1)~(3)||--
|-
|-
-
|8/29 Plux-RBS-GFP-DT(8/27 LB-CP) (1)~(2)||1227
+
|9/13 Plux＋RBS-lysis2-DT (1)~(3)||-
|-
|-
-
|8/29 Pbad/araC-RBS-RFP{BBa_I13516} (8/21 LB-CP) (1)~(2)||2256
+
|9/13 Plux＋RBS-lysis3-DT (1)~(2)||-
 +
|}
 +
{| class="wikitable"
 +
!Sample||base pair
|-
|-
-
|8/29 RBS-lysis3-DT-(1)||1210
+
|9/13 Pcon-RBS-luxR-DT＋Plux-RBS-GFP-DT (1)~(4)||--
 +
|-
 +
|9/13 Pcon-PT181attenuator＋aptamer121R-DT (1)~(2)||-
 +
|-
 +
|9/13 Plac＋PT181attenuator (1)||-
|}
|}
{| class="wikitable"
{| class="wikitable"
-
!PreDenature||Denature||Annealing||Extension||cycle
+
!Sample||base pair
|-
|-
-
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
+
|Dλ-lux(1) (1)~(2)||--
|-
|-
-
|5min||30s||30s||2min||30cycle
+
|NC ||--
|}
|}
</div>
</div>
-
===Liquid Culture===
+
===Electrophoresis===
-
<!-- ここから -->
+
<div class="experiment">
<div class="experiment">
<span class="author">No name</span>
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
-
!Sample||medium
+
!Lane||Sample
|-
|-
-
|8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1)||Plusgrow medium(+Amp)
+
|1||1kbp ladder
|-
|-
-
|8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(2)||Plusgrow medium(+Amp)
+
|2||9/13 Plux+RBS-lysis1-DT1
|-
|-
-
|8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(3)||Plusgrow medium(+Amp)
+
|3||9/13 Plux+RBS-lysis1-DT2
|-
|-
-
|8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(4)||Plusgrow medium(+Amp)
+
|4||9/13 Plux+RBS-lysis1-DT3
|-
|-
-
|8/29 Plux-RBS-GFP-DT-(1)||Plusgrow medium(+CP)
+
|5||9/13 Plux+RBS-lysis2-DT1
|-
|-
-
|8/29 Plux-RBS-GFP-DT-(2)||Plusgrow medium(+CP)
+
|6||9/13 Plux+RBS-lysis2-DT2
|-
|-
-
|8/29 Pbad/araC-RBS-RFP-DT-(1)||Plusgrow medium(+CP)
+
|7||9/13 Plux+RBS-lysis2-DT3
|-
|-
-
|8/29 Pbad-araC-RBS-RFP-DT-(2)||Plusgrow medium(+CP)
+
|8||9/13 Plux+RBS-lysis3-DT1
|-
|-
-
|RBS-lysis3-DT-(1)||Plusgrow medium(+CP)
+
|9||9/13 Plux+RBS-lysis3-DT2
 +
|-
 +
|10||9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT1
 +
|-
 +
|11||9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT2
 +
|-
 +
|12||9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT3
 +
|-
 +
|13||9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT4
 +
|-
 +
|14||9/13 Ptet-pT181 antisense+Spinach-DT1
 +
|-
 +
|15||9/13 Pcon-pT181 attenuator+aptamer12-1R-PT1
 +
|-
 +
|16||9/13 Pcon-pT181 attenuator+aptamer12-1R-PT1
 +
|-
 +
|17||9/13 Plac+pT181 attenuator
 +
|}
 +
[[File:Igku_0914_E5.jpg]]<br>
 +
</div>
 +
 
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1||1kbp ladder
 +
|-
 +
|2||P&lambda;-lux(1)1
 +
|-
 +
|3|| P&lambda;-lux(1)1
 +
|-
 +
|4||NC
 +
|}
 +
[[File:Igku_0914_E6.jpg]]<br>
 +
 
 +
===Liquid Culture===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Sample||medium
 +
|-
 +
|9/13 Pλ-luxI(1)-1||Plusgrow medium(+Amp)
|}
|}
</div>
</div>
Line 225: Line 291:
===Transformation===
===Transformation===
<div class="experiment">
<div class="experiment">
-
<span class="author">No Name</span>
+
<span class="author">Nakamoto</span>
{| class="wikitable"
{| class="wikitable"
!Name||Sample||Competent Cells||Total||Plate
!Name||Sample||Competent Cells||Total||Plate
|-
|-
-
|8/27 pT181 attenuator-DT||2&micro;L||20&micro;L||22&micro;L||CP
+
|9/13 RBS-lysisZα-DT(XbaI&PstI)＋9/13 Pcon-PT181 attenuator(SpeI&PstI)||2&micro;L||20&micro;L||22&micro;L||Amp
|-
|-
-
|8/27 RBS-lysis3-DT||2&micro;L||20&micro;L||22&micro;L||CP
+
|9/13 SB2C3＋9/14 PzαB||2&micro;L||20&micro;L||22&micro;L||CP
|-
|-
-
|8/28 RBS-lysis3-DT||2&micro;L||20&micro;L||22&micro;L||CP
+
|9/13 pSB2C3＋9/14 PkaiBC||2&micro;L||20&micro;L||22&micro;L||CP
|-
|-
-
|8/28 Pcon-RBS-GFP-DT-Pcon-RBS-LuxR-DT||2&micro;L||20&micro;L||22&micro;L||Amp
+
|9/13 RBS-GFP-DT＋9/14 PkaiBC||2&micro;L||20&micro;L||22&micro;L||CP
|-
|-
-
|8/28 RBS-lysis1-DT||2&micro;L||20&micro;L||22&micro;L||CP
+
|Plac＋RBS-lysis1-DT||2&micro;L||20&micro;L||22&micro;L||Amp
|-
|-
-
|8/28 Plux-RBS-GFP-DT||2&micro;L||20&micro;L||22&micro;L||CP
+
|Plac＋RBS-lysis2-DT||2&micro;L||20&micro;L||22&micro;L||Amp
|-
|-
-
|8/28 Spinach-DT||2&micro;L||20&micro;L||22&micro;L||CP
+
|Plac＋RBS-lysis2-DT||2&micro;L||20&micro;L||22&micro;L||Amp
|}
|}
-
[[File:Igku Aug29 Transformation1(N④-1).jpg]]<br>
 
-
[[File:Igku Aug29 Transformation(N④-2).jpg]]<br>
 
-
[[File:Igku Aug29 Transformation(N⑤).jpg]]<br>
 
-
[[File:Igku Aug29 Transformation()after]]<br>
 
</div>
</div>
-
 
+
===Sequence PCR===
-
===PCR===
+
<div class="experiment">
<div class="experiment">
-
<span class="author">No name</span>
+
<span class="author">Tatsui</span>
{| class="wikitable"
{| class="wikitable"
-
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||SasA_fwd primer||SasA_rev primer||MilliQ||total
+
!genome DNA||temp||Big Dye||5x buffer||primer||MilliQ
 +
|-
 +
|9/6 PT181 attenuator(VR)||3.9||0.5||1.75||0.5||3.85
 +
|-
 +
|8/17 RBS lacZα-DT||2.1||0.5||1.75||0.5||5.65
|-
|-
-
| ||0.5||2.5||2.5||1.5||0.75||0.75|| ||25
+
|9/6 PT181 attenuator(VR)||3.9||0.5||1.75||0.5||3.85
|}
|}
{| class="wikitable"
{| class="wikitable"
!PreDenature||Denature||Annealing||Extension||cycle
!PreDenature||Denature||Annealing||Extension||cycle
|-
|-
-
|　&deg;C||　&deg;C||　&deg;C||　&deg;C||--
+
|96&deg;C||50&deg;C||60&deg;C||4&deg;C||--
|-
|-
-
|（◎Д◎）　||　||　||　||
+
|10sec||5sec||4min||1min||30cycle
|}
|}
</div>
</div>

Latest revision as of 17:41, 27 September 2013

Contents

Sep 14

Restriction Enzyme Digestion

Hirano

9/14 pSB4K5SEcoRIXbaISpeIPstIBufferBBufferDBSAMilliQtotal
2cuts10µL1µL0µL1µL0µL3µL0µL3µL12µL30µL
NC0.5µL0µL0µL0µL0µL1µL0µL1µL7.5µL10µL
9/13 PlacXbaIPstIBufferHBSAMilliQtotal
1 cut9µL0µL1µL3µL3µL14µL30µL
NC1µL0µL0µL1µL1µL7µL10µL
9/14 PKaiBCEcoRIXbaISpeIPstIBufferBBSAMilliQtotal
2cuts11µL1µL0µL1µL0µL3µL3µL11µL30µL
NC4µL0µL0µL0µL0µL1µL1µL4µL10µL
9/14 RpaBEcoRIXbaISpeIPstIBufferDBSAMilliQtotal
2cuts13µL0µL1µL0µL1µL3µL3µL9µL30µL
NC2µL0µL0µL0µL0µL1µL1µL5µL10µL
8/17 RBS-GFP-DTEcoRIXbaISpeIPstIBufferDBSAMilliQtotal
1cut9µL0µL1µL0µL0µL3µL3µL14µL30µL
NC0.5µL0µL0µL0µL0µL1µL1µL7.5µL10µL
  • incubated 37°C 1hour

Liquid Culture

Hirano

Samplemedium
9/11 Plac(BBa-R0011)-(3)Plusgrow medium(+Amp)

Electrophoresis

Hirano

LaneSampleEnzyme1Enzyme2
11kb ladder----
2pSB4K5(1)EcoRISpeI
3 pSB4K5(1)----
4Plac(1A2)PstI--
5Plac(1A2)----
6PKaiBCEcoRISpeI
7PKaiBC----
8RpaB(1)XbaIPstI
9RpaB(1)----
10RBS-GFP-DTXbaI--
11RBS-GFP-DT----
121kb ladder----

Igku 0914 E1.jpg

LaneSampleEnzyme
11kb ladder--
2Plac(pSB1A2)PstI
3Plac(pSB1A2)--

Igku 0914 E2.jpg

Sequence PCR

No name

genome DNABig Dye5x buffertemp(400mg)primermilliQ
Pcon-RBS-luxR-DT0.51.750.90.56.85
Pcon-RBS-luxR-DT0.51.750.40.57.35
Pcon-RBS-tetR-DT0.51.7530.55.25
Pcon-RBS-tetR-DT0.51.7530.55.25
Plux0.51.752.40.55.35
Plux-RBS-GFP-DT0.51.751.90.55.85
Plux-RBS-GFP-DT0.50.51.90.55.85
Pcon-RBS-GFP-DT0.50.51.20.56.55
Pcon-RBS-GFP-DT0.50.51.20.56.55
RBS-lysis3-DT0.50.51.00.56.75
Pcon-antisense-Spinach-DT0.50.51.40.56.35
Pcon-RBS lacZα-DT0.50.51.40.56.35
Ptet-RBS lacZα-DT0.50.50.590.51.85
PreDenatureDenatureAnnealingExtensioncycle
96°C96°C50°C60°C--
2min10sec5sec4min30cycle

Gel Extraction

Tatsui

LaneDNAEnzyme
11kb ladder--
2pSB4K5EcoRI&SpeI
3pSB4K5EcoRI&SpeI
4pSB4K5EcoRI&SpeI

Igku 0914 G8.jpg
Igku 0914 G9.jpg

Restriction Enzyme Digestion

No name

9/14 Plac(1A2)EcoRIXbaISpeIPstIBufferBBSAMilliQtotal
2cuts23µL0µL0µL0.3µL0µL3µL3µL0µL30µL
9/14 RBS-GFP-DTEcoRIXbaISpeIPstIBufferHBSAMilliQtotal
2 cuts23µL1µL0µL0µL0µL3µL3µL0µL30µL

Ligation

Nakamoto and Hirano

stateVectorInserterLigation High ver.2
experiment9/13 Pcon-PT181attenuator(SpeI&PstI)1.79/13 RBS-lacZα-DT (XbaI & PstI)12.55
experiment9/13 pSB2C3 (XbaI & PstI)1.99/14 RpaB (XbaI & PstI)16.310.5
experiment9/13 pSB2C3 (XbaI & PstI)4.79/14 PkaiBC (EcoRI & SpeI)6.75.7
experiment9/14 RBS-GFP-DT (PstI&XbaI)0.79/14 PkaiBC (EcoRI & SpeI)2.61.6
experiment9/14 Plac (SpeI&PstI)2.49/14 RBS-lysis1-DT (XbaI & PstI)4.93.6
experiment9/14 Plac (SpeI&PstI)2.49/14 RBS-lysis2-DT (XbaI & PstI)7.04.7
experiment9/14 Plac (SpeI&PstI)2.49/14 RBS-lysis3-DT (XbaI & PstI)9.25.8

Samples were evaporeted used evaporator into about 7 µL

Colony PCR

No name

Samplebase pair
9/13 Plux＋RBS-lysis1-DT (1)~(3)--
9/13 Plux＋RBS-lysis2-DT (1)~(3)-
9/13 Plux＋RBS-lysis3-DT (1)~(2)-
Samplebase pair
9/13 Pcon-RBS-luxR-DT＋Plux-RBS-GFP-DT (1)~(4)--
9/13 Pcon-PT181attenuator＋aptamer121R-DT (1)~(2)-
9/13 Plac＋PT181attenuator (1)-
Samplebase pair
Dλ-lux(1) (1)~(2)--
NC --

Electrophoresis

No name

LaneSample
11kbp ladder
29/13 Plux+RBS-lysis1-DT1
39/13 Plux+RBS-lysis1-DT2
49/13 Plux+RBS-lysis1-DT3
59/13 Plux+RBS-lysis2-DT1
69/13 Plux+RBS-lysis2-DT2
79/13 Plux+RBS-lysis2-DT3
89/13 Plux+RBS-lysis3-DT1
99/13 Plux+RBS-lysis3-DT2
109/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT1
119/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT2
129/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT3
139/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT4
149/13 Ptet-pT181 antisense+Spinach-DT1
159/13 Pcon-pT181 attenuator+aptamer12-1R-PT1
169/13 Pcon-pT181 attenuator+aptamer12-1R-PT1
179/13 Plac+pT181 attenuator

Igku 0914 E5.jpg

LaneSample
11kbp ladder
2Pλ-lux(1)1
3 Pλ-lux(1)1
4NC

Igku 0914 E6.jpg

Liquid Culture

No name

Samplemedium
9/13 Pλ-luxI(1)-1Plusgrow medium(+Amp)

Transformation

Nakamoto

NameSampleCompetent CellsTotalPlate
9/13 RBS-lysisZα-DT(XbaI&PstI)＋9/13 Pcon-PT181 attenuator(SpeI&PstI)2µL20µL22µLAmp
9/13 SB2C3＋9/14 PzαB2µL20µL22µLCP
9/13 pSB2C3＋9/14 PkaiBC2µL20µL22µLCP
9/13 RBS-GFP-DT＋9/14 PkaiBC2µL20µL22µLCP
Plac＋RBS-lysis1-DT2µL20µL22µLAmp
Plac＋RBS-lysis2-DT2µL20µL22µLAmp
Plac＋RBS-lysis2-DT2µL20µL22µLAmp

Sequence PCR

Tatsui

genome DNAtempBig Dye5x bufferprimerMilliQ
9/6 PT181 attenuator(VR)3.90.51.750.53.85
8/17 RBS lacZα-DT2.10.51.750.55.65
9/6 PT181 attenuator(VR)3.90.51.750.53.85
PreDenatureDenatureAnnealingExtensioncycle
96°C50°C60°C4°C--
10sec5sec4min1min30cycle
Retrieved from "http://2013.igem.org/Template:Kyoto/Notebook/Sep_14"