Template:Kyoto/Notebook/Sep 11

From 2013.igem.org

(Difference between revisions)
(→colony PCR)
(→Restriction Enzyme Digestion)
 
(65 intermediate revisions not shown)
Line 1: Line 1:
==Sep 11==
==Sep 11==
 +
===Electrophoresis===
 +
 +
<div class="experiment">
 +
<span class="author"></span>
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1||1kb ladder
 +
|-
 +
|2||Pbad/araC+RBS-luxI-DT-1
 +
|-
 +
|3||Pbad/araC+RBS-luxI-DT-1 
 +
|-
 +
|4||Plac+RBS-lysis-DT
 +
|-
 +
|5||Pcon+RBS-luxI-DT
 +
|-
 +
|6||1kb ladder 
 +
|}
 +
[[File:igku_9111.jpg]]<br>
 +
</div>
 +
===PCR===
===PCR===
<div class="experiment">
<div class="experiment">
Line 6: Line 28:
!pKaiBC(200p)||10*buffer KOD plus||dNTPs||MgSO4||primer mix(F&R)||KOD plus||MilliQ||total
!pKaiBC(200p)||10*buffer KOD plus||dNTPs||MgSO4||primer mix(F&R)||KOD plus||MilliQ||total
|-
|-
-
|0.5||2.5||2.5||1.5||1.5||0.5||16|| ||25
+
|0.5||2.5||2.5||1.5||1.5||0.5||16||25
|}
|}
{| class="wikitable"
{| class="wikitable"
Line 20: Line 42:
!pKaiBC(400p)||10*buffer KOD plus||dNTPs||MgSO4||primer mix(F&R)||KOD plus||MilliQ||total
!pKaiBC(400p)||10*buffer KOD plus||dNTPs||MgSO4||primer mix(F&R)||KOD plus||MilliQ||total
|-
|-
-
|1||2.5||2.5||1.5||1.5||0.5||15.5|| ||25
+
|1||2.5||2.5||1.5||1.5||0.5||15.5||25
|}
|}
{| class="wikitable"
{| class="wikitable"
Line 34: Line 56:
!pKaiBC(800p)||10*buffer KOD plus||dNTPs||MgSO4||primer mix(F&R)||KOD plus||MilliQ||total
!pKaiBC(800p)||10*buffer KOD plus||dNTPs||MgSO4||primer mix(F&R)||KOD plus||MilliQ||total
|-
|-
-
|2||2.5||2.5||1.5||1.5||0.5||14.5|| ||25
+
|2||2.5||2.5||1.5||1.5||0.5||14.5||25
|}
|}
{| class="wikitable"
{| class="wikitable"
Line 49: Line 71:
!pKaiBC(1ng)||10*buffer KOD plus||dNTPs||MgSO4||primer mix(F&R)||KOD plus||MilliQ||total
!pKaiBC(1ng)||10*buffer KOD plus||dNTPs||MgSO4||primer mix(F&R)||KOD plus||MilliQ||total
|-
|-
-
|2.5||2.5||2.5||1.5||1.5||0.5||14|| ||25
+
|2.5||2.5||2.5||1.5||1.5||0.5||14||25
|}
|}
{| class="wikitable"
{| class="wikitable"
Line 59: Line 81:
|}
|}
</div>
</div>
 +
===Electrophoresis===
 +
 +
<!-- こっから -->
 +
<div class="experiment">
 +
<span class="author">Tatsui</span>
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1||100bp ladder 
 +
|-
 +
|2||pKaiBC 800pg 60&deg;C
 +
|-
 +
|3||pKaiBC 1ng 60&deg;C 
 +
|-
 +
|4||pKaiBC 800pg 61&deg;C
 +
|-
 +
|5||pKaiBC 1ng 61&deg;C
 +
|-
 +
|6||pKaiBC 800pg 62&deg;C
 +
|-
 +
|7||pKaiBC 1ng 62&deg;C 
 +
|-
 +
|8||100bp ladder
 +
|}
 +
[[File:igku_9112.jpg]]<br>
 +
</div>
 +
<!-- ここまでをコピーしてね -->
 +
===colony PCR===
===colony PCR===
<div class="experiment">
<div class="experiment">
Line 66: Line 116:
|-
|-
|9/9Pcon-lacZ&alpha;(p3B4k5)||712bp
|9/9Pcon-lacZ&alpha;(p3B4k5)||712bp
 +
|-
 +
|9/9Pcon-spinach-DT(p3B4k5)||605bp
|}
|}
 +
 +
</div>
 +
 +
<div class="experiment">
 +
<span class="author">Tatsui</span>
{| class="wikitable"
{| class="wikitable"
!PreDenature||Denature||Annealing||Extension||cycle
!PreDenature||Denature||Annealing||Extension||cycle
Line 72: Line 129:
|　94&deg;C||　94&deg;C||　55&deg;C||　68&deg;C||--
|　94&deg;C||　94&deg;C||　55&deg;C||　68&deg;C||--
|-
|-
-
|5min||30s　||30s　||42s　||
+
|5min||30s　||30s　||42s　||30
|}
|}
</div>
</div>
 +
 +
<div>
<div>
{| class="wikitable"
{| class="wikitable"
!Sample||base pair
!Sample||base pair
|-
|-
-
|9/9Pcon-spinach-DT(p3B4k5)||605bp
+
|Pbad\araC-RBS-luxI-DT||
|}
|}
{| class="wikitable"
{| class="wikitable"
Line 86: Line 145:
|　94&deg;C||　94&deg;C||　55&deg;C||　68&deg;C||--
|　94&deg;C||　94&deg;C||　55&deg;C||　68&deg;C||--
|-
|-
-
|5min||30s　||30s　||42s　||
+
|5min||30s　||30s　||2min　||30
|}
|}
</div>
</div>
-
<div>
+
 
 +
===Electrophoresis===
 +
 +
<!-- こっから -->
 +
<div class="experiment">
 +
<span class="author">Tatsui</span>
{| class="wikitable"
{| class="wikitable"
-
!Sample||base pair
+
!Lane||Sample
|-
|-
-
|Pbad\araC-RBS-luxI-DT||
+
|1||100bp ladder 
 +
|-
 +
|2||Pcon-lacZ&alpha;(pSB4K5)3
 +
|-
 +
|3||Pcon-lacZ&alpha;(pSB4K5)4 
 +
|-
 +
|4||Pcon-lacZ&alpha;(pSB4K5)5
 +
|-
 +
|5||Pcon-lacZ&alpha;(pSB4K5)6
 +
|-
 +
|6||Pcon-spinach-DT(pSB4K5)3
 +
|-
 +
|7||Pcon-spinach-DT(pSB4K5)4 
 +
|-
 +
|8||Pcon-spinach-DT(pSB4K5)5 
 +
|-
 +
|9||Pcon-spinach-DT(pSB4K5)6 
 +
|-
 +
|10||100bp ladder
|}
|}
 +
[[File:igku_9113.jpg]]<br>
{| class="wikitable"
{| class="wikitable"
-
!PreDenature||Denature||Annealing||Extension||cycle
+
!Lane||Sample
|-
|-
-
|　94&deg;C||　94&deg;C||　55&deg;C||　68&deg;C||--
+
|1||1kbp ladder 
|-
|-
-
|5min||30s　||30s　||2min　||
+
|2||Pbad/araC-RBS-luxI-DT3
 +
|-
 +
|3||Pbad/araC-RBS-luxI-DT4 
 +
|-
 +
|4||Pbad/araC-RBS-luxI-DT5
 +
|-
 +
|5||Pbad/araC-RBS-luxI-DT6
 +
|-
 +
|6||1kbp ladder
|}
|}
 +
[[File:igku_9114.jpg]]<br>
</div>
</div>
 +
 +
===colony PCR===
<div class="experiment">
<div class="experiment">
{| class="wikitable"
{| class="wikitable"
Line 108: Line 202:
|-
|-
|9/9Pcon+RBS-luxI-DT||1079bp
|9/9Pcon+RBS-luxI-DT||1079bp
 +
|-
 +
|9/10Pcon+sptamer12_1R-DT||584bp
 +
|-
 +
|9/10DT+Pcon-pT181 attenuator 1||781bp
 +
|-
 +
|9/10Fusion1 attenuator(p3B1C3)-1||638bp
|}
|}
{| class="wikitable"
{| class="wikitable"
Line 114: Line 214:
|　94&deg;C||　94&deg;C||　55&deg;C||　68&deg;C||--
|　94&deg;C||　94&deg;C||　55&deg;C||　68&deg;C||--
|-
|-
-
|50sec　||30sec　||30sec　||1min　||
+
|50sec　||30sec　||30sec　||1min　||30
|}
|}
</div>
</div>
 +
 +
===Gel Extraction===
 +
<!-- こっから -->
 +
<div class="experiment">
 +
<span class="author"></span>
 +
[[File:igku_9115.jpg]]<br>
 +
[[File:igku_9116.jpg]]<br>
 +
[[File:igku_9117.jpg]]<br>
 +
[[File:igku_9118.jpg]]<br>
 +
</div>
 +
<!-- ここまでをコピーしてね -->
 +
 +
===Ligation===
<div class="experiment">
<div class="experiment">
 +
<span class="author">Okazaki</span>
{| class="wikitable"
{| class="wikitable"
-
!Sample||base pair
+
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
|-
|-
-
|9/10Pcon+sptamer12_1R-DT||584bp
+
|experiment||9/8pSB4K5(EcoRI&SpeI)21.8ng/&micro;L||2.3 &micro;L||9/8Pcon-Spinach-DT(EcoRI&SpeI)10.5ng/&micro;L||1.7 &micro;L||2.0 &micro;L
 +
|-
 +
|experment||9/8pSB4K5(EcoRI&SpeI)21.8ng/&micro;L||2.3 &micro;L||9/8RBS-lacZ&alpha;-DT(EcoRI&SpeI)4.4ng/&micro;L|| 6.7&micro;L||3.5 &micro;L
 +
|-
 +
|experiment||9/8pSB4K5(EcoRI&SpeI)21.8ng/&micro;L||2.3 &micro;L||9/8Pcon-RBS-lacZ&alpha;-DT(EcoRI&SpeI)5.6ng/&micro;L||1.9 &micro;L||2.1 &micro;L
 +
|-
 +
|experiment||9/6Plux 25ng/&micro;L||2 &micro;L||9/8 RBS-lysis1-DT(XbaI&PstI)||6.6 &micro;L||3.5 &micro;L
 +
|-
 +
|experiment||9/6Plux 25ng/&micro;L||2 &micro;L||9/8 RBS-lysis3-DT(XbaI&PstI)||4.7 &micro;L||3.4 &micro;L
 +
|-
 +
|experiment||9/10Pcon-pT181 attenuator(SpeI&PstI)|| 3.4&micro;L||9/8RBS-lacZ&alpha;-DT(EcoRI&SpeI)||4.5 &micro;L||3.5 &micro;L
 +
|-
 +
|experiment||9/6Pcon-RBS laxR-DT(EcoRI&XbaI) 26.7ng/&micro;L||1.9 &micro;L||Plax-RBS-GFP-DT||5.5 &micro;L||3.5 &micro;L
 +
|-
 +
|experiment||9/9 Pcon-pT181 attenuator(SpeI&PstI)||3.4 &micro;L||9/10 aptamer12_1R-DT(XbaI&PstI)||1.7 &micro;L||2.6 &micro;L
|}
|}
 +
</div>
 +
 +
===Transformation===
 +
<div class="experiment">
 +
<span class="author">Nakamoto Tatsui</span>
{| class="wikitable"
{| class="wikitable"
-
!PreDenature||Denature||Annealing||Extension||cycle
+
!Name||Sample||Competent Cells||Total||Plate
|-
|-
-
|　94&deg;C||　94&deg;C||　55&deg;C||　68&deg;C||--
+
|9/9 Fusion1 antisense(pSB2C3)||2&micro;L||20&micro;L||22&micro;L||CP
|-
|-
-
|50sec　||30sec　||30sec　||1min　||
+
|9/9 Fusion6 antisense(pSB2C3)||2&micro;L||20&micro;L||22&micro;L||CP
 +
|-
 +
|9/9 aptamer 12_P(pSB2C3)||2&micro;L||20&micro;L||22&micro;L||CP
 +
|-
 +
|9/9 aptamer 12_1&micro(pSB2C3)||2&micro;L||20&micro;L||22&micro;L||CP
 +
|-
 +
|9/9 Fusion2 attenuator(pSB2C3) ||2&micro;L||20&micro;L||22&micro;L||CP
 +
|-
 +
|9/11 Pcon-spinach-DT(pSB4K5)||2&micro;L||20&micro;L||22&micro;L||K
 +
|-
 +
|9/11 RBS-lacZ&alpha;-DT(pSB4K5)||2&micro;L||20&micro;L||22&micro;L||K
 +
|-
 +
|9/11 Pcon-RBS-lacZ&alpha;-DT(pSB4K5)||2&micro;L||20&micro;L||22&micro;L||K
 +
|-
 +
|Plux+RBS-lysis1-DT||2&micro;L||20&micro;L||22&micro;L||CP
 +
|-
 +
|Plux+RBS-lysis3-DT||2&micro;L||20&micro;L||22&micro;L||CP
 +
|-
 +
|Pcon-pT181attenuator+RBS-lacZ&alpha;-DT||2&micro;L||20&micro;L||22&micro;L||Amp
 +
|-
 +
|Pcon-iuxR+Plux-GFP||2&micro;L||20&micro;L||22&micro;L||Amp
 +
|-
 +
|Pcon-pT181attenuator+apt12_1R-DT||2&micro;L||20&micro;L||22&micro;L||Amp
 +
|-
 +
|Plac(BBa_R0011)||2&micro;L||20&micro;L||22&micro;L||Amp||
|}
|}
</div>
</div>
 +
 +
===Electrophoresis===
 +
 +
<!-- こっから -->
<div class="experiment">
<div class="experiment">
 +
<span class="author">Tatsui</span>
{| class="wikitable"
{| class="wikitable"
-
!Sample||base pair
+
!Lane||Sample
|-
|-
-
|9/10DT+Pcon-pT181 attenuater 1||781bp
+
|1||100bp ladder 
 +
|-
 +
|2||Pcon+RBS luxI-DT2
 +
|-
 +
|3||Pcon+RBS luxI-DT3 
 +
|-
 +
|4||Pcon+RBS luxI-DT4
 +
|-
 +
|5||Pcon+RBS luxI-DT5 
 +
|-
 +
|6||Pcon+RBS luxI-DT6 
 +
|-
 +
|7||Pcon+apF12_1R-DT1
 +
|-
 +
|8||Pcon+apF12_1R-DT2
 +
|-
 +
|9||Pcon+apF12_1R-DT3 
 +
|-
 +
|10||Pcon+apF12_1R-DT4
 +
|-
 +
|11||DT+Pcon-pT181attenuator1
 +
|-
 +
|12||F1attenuator(pSB1C3)
 +
|-
 +
|13||100bp ladder
|}
|}
 +
[[File:igku_9119.jpg]]<br>
 +
</div>
 +
 +
===Restriction Enzyme Digestion===
 +
<div class="experiment">
 +
<span class="author">Nakamoto</span>
{| class="wikitable"
{| class="wikitable"
-
!PreDenature||Denature||Annealing||Extension||cycle
+
! ||DNA||EcoRI||XbaI||SpeI||PstI||buffer||BSA||MilliQ||Total
|-
|-
-
|　94&deg;C||　94&deg;C||　55&deg;C||　68&deg;C||--
+
|pSB4K5(EcoRI+SpeI)||9.4&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 3&micro;L|| 3&micro;L|| 12.6&micro;L||30 &micro;L
|-
|-
-
|50sec　||30sec　||30sec　||1min　||
+
|pSB4K5(EcoRI+SpeI) negative||0.5&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 7.5&micro;L|| 10&micro;L
 +
|-
 +
|RBS-lacZ&alpha;-DT(EcoRI+SpeI)|| 10&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 3&micro;L|| 3&micro;L|| 12&micro;L|| 30&micro;L
 +
|-
 +
|RBS-lacZ&alpha;-DT(EcoRI+SpeI) negative|| 0.5&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 7.5&micro;L|| 10&micro;L
 +
|-
 +
|Ptet-pT181 antisense(SteI+PstI)|| 10.8&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L|| 12&micro;L|| 30&micro;L
 +
|-
 +
|Ptet-pT181 antisense(SteI+PstI) negative|| 0.5&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 7.5&micro;L|| 10&micro;L
 +
|-
 +
|Pcon-RBS-lacZ&alpha;-DT(EcoRI+SpeI)|| 7.1&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 3&micro;L|| 3&micro;L|| 14.9&micro;L|| 30&micro;L
 +
|-
 +
|Pcon-RBS-lacZ&alpha;-DT(EcoRI+SpeI) negative|| 0.4&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 7.6&micro;L|| 10&micro;L
 +
|-
 +
|RBS-lysis2-DT(XbaI+PstI)|| 22&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L|| 0&micro;L|| 30&micro;L
 +
|-
 +
|RBS-lysis2-DT(XbaI+PstI) negative|| 1.4&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 6.6&micro;L|| 10&micro;L
|}
|}
</div>
</div>
 +
 +
===Liquid Culture===
<div class="experiment">
<div class="experiment">
 +
<span class="author">Nakamoto</span>
{| class="wikitable"
{| class="wikitable"
-
!Sample||base pair
+
!Sample||medium
|-
|-
-
|9/10Fusion1 attenuater(p3B1C3)-1||638bp
+
|8/16 Master4 Plax||
 +
|-
 +
|8/25 P&lambda;-laxI-2 ||　
 +
|}
 +
</div>
 +
 
 +
===PCR===
 +
<div class="experiment">
 +
<span class="author">Hirano</span>
 +
{| class="wikitable"
 +
!8/29 P&lambda;-RBS-luxI-DT(76.5ng/&micro;L)||10x buffer for KOD plus-ver.2||KOD-plus||dNTP||MgSO4||F primer(RBS-luxI-DT-cloning-fwd)||R primer(RBS-luxI-DT-cloning-rev)||MilliQ||total
 +
|-
 +
|0.3||2.5||0.5||2.5||1.5||0.75||0.75||16.2||25
|}
|}
{| 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||　65&deg;C||　68&deg;C||--
 +
|-
 +
|5min||30sec||30sec||1min||30
 +
|}
 +
 
 +
<div class="experiment">
 +
{| class="wikitable"
 +
!8/31 Pbad/araC-RBS-RFP(1)(67.6ng/&micro;L)||10x buffer for KOD plus-ver.2||KOD-plus||dNTP||MgSO4||F primer(Pbad/araC(pSBlc3)-cloning-fwd)||R primer(Pbad/araC(pSBlc3)-cloning-rev)||MilliQ||total
 +
|-
 +
|0.3||2.5||0.5||2.5||1.5||0.75||0.75||16.2||25
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|　94&deg;C||　94&deg;C||　54&deg;C||　68&deg;C||--
 +
|-
 +
|5min||30sec||30sec||3min 20sec||30
 +
|}
 +
 
 +
===Liquid Culture===
 +
<div class="experiment">
 +
<span class="author">Hirano</span>
 +
{| class="wikitable"
 +
!Sample||medium
 +
|-
 +
|9/4 RBS-lysis1+DT 3||plusgrow(+CP) 3ml 37&deg;C
 +
|-
 +
|9/4 RBS-lysis2+DT 1||plusgrow(+CP) 3ml 37&deg;C
|-
|-
-
|50sec　||30sec　||30sec　||1min　||
+
|9/4 RBS-lysis3+DT 3||plusgrow(+CP) 3ml 37&deg;C
|}
|}
</div>
</div>

Latest revision as of 15:00, 27 September 2013

Contents

Sep 11

Electrophoresis

LaneSample
11kb ladder
2Pbad/araC+RBS-luxI-DT-1
3Pbad/araC+RBS-luxI-DT-1
4Plac+RBS-lysis-DT
5Pcon+RBS-luxI-DT
61kb ladder

Igku 9111.jpg

PCR

Tatsui

pKaiBC(200p)10*buffer KOD plusdNTPsMgSO4primer mix(F&R)KOD plusMilliQtotal
0.52.52.51.51.50.51625
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　60°C　68°C30
2min　10sec　30sec　30sec　
pKaiBC(400p)10*buffer KOD plusdNTPsMgSO4primer mix(F&R)KOD plusMilliQtotal
12.52.51.51.50.515.525
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　60°C　68°C30
2min　10sec　30sec　30sec　
pKaiBC(800p)10*buffer KOD plusdNTPsMgSO4primer mix(F&R)KOD plusMilliQtotal
22.52.51.51.50.514.525
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　60°C　68°C30
2min　10sec　30sec　30sec　
pKaiBC(1ng)10*buffer KOD plusdNTPsMgSO4primer mix(F&R)KOD plusMilliQtotal
2.52.52.51.51.50.51425
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　60°C　68°C30
2min　10sec　30sec　30sec　

Electrophoresis

Tatsui

LaneSample
1100bp ladder
2pKaiBC 800pg 60°C
3pKaiBC 1ng 60°C
4pKaiBC 800pg 61°C
5pKaiBC 1ng 61°C
6pKaiBC 800pg 62°C
7pKaiBC 1ng 62°C
8100bp ladder

Igku 9112.jpg

colony PCR

Tatsui

Samplebase pair
9/9Pcon-lacZα(p3B4k5)712bp
9/9Pcon-spinach-DT(p3B4k5)605bp

Tatsui

PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
5min30s　30s　42s　30


Samplebase pair
Pbad\araC-RBS-luxI-DT
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
5min30s　30s　2min　30

Electrophoresis

Tatsui

LaneSample
1100bp ladder
2Pcon-lacZα(pSB4K5)3
3Pcon-lacZα(pSB4K5)4
4Pcon-lacZα(pSB4K5)5
5Pcon-lacZα(pSB4K5)6
6Pcon-spinach-DT(pSB4K5)3
7Pcon-spinach-DT(pSB4K5)4
8Pcon-spinach-DT(pSB4K5)5
9Pcon-spinach-DT(pSB4K5)6
10100bp ladder

Igku 9113.jpg

LaneSample
11kbp ladder
2Pbad/araC-RBS-luxI-DT3
3Pbad/araC-RBS-luxI-DT4
4Pbad/araC-RBS-luxI-DT5
5Pbad/araC-RBS-luxI-DT6
61kbp ladder

Igku 9114.jpg

colony PCR

Samplebase pair
9/9Pcon+RBS-luxI-DT1079bp
9/10Pcon+sptamer12_1R-DT584bp
9/10DT+Pcon-pT181 attenuator 1781bp
9/10Fusion1 attenuator(p3B1C3)-1638bp
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
50sec　30sec　30sec　1min　30

Gel Extraction

Igku 9115.jpg
Igku 9116.jpg
Igku 9117.jpg
Igku 9118.jpg

Ligation

Okazaki

stateVectorInserterLigation High ver.2
experiment9/8pSB4K5(EcoRI&SpeI)21.8ng/µL2.3 µL9/8Pcon-Spinach-DT(EcoRI&SpeI)10.5ng/µL1.7 µL2.0 µL
experment9/8pSB4K5(EcoRI&SpeI)21.8ng/µL2.3 µL9/8RBS-lacZα-DT(EcoRI&SpeI)4.4ng/µL 6.7µL3.5 µL
experiment9/8pSB4K5(EcoRI&SpeI)21.8ng/µL2.3 µL9/8Pcon-RBS-lacZα-DT(EcoRI&SpeI)5.6ng/µL1.9 µL2.1 µL
experiment9/6Plux 25ng/µL2 µL9/8 RBS-lysis1-DT(XbaI&PstI)6.6 µL3.5 µL
experiment9/6Plux 25ng/µL2 µL9/8 RBS-lysis3-DT(XbaI&PstI)4.7 µL3.4 µL
experiment9/10Pcon-pT181 attenuator(SpeI&PstI) 3.4µL9/8RBS-lacZα-DT(EcoRI&SpeI)4.5 µL3.5 µL
experiment9/6Pcon-RBS laxR-DT(EcoRI&XbaI) 26.7ng/µL1.9 µLPlax-RBS-GFP-DT5.5 µL3.5 µL
experiment9/9 Pcon-pT181 attenuator(SpeI&PstI)3.4 µL9/10 aptamer12_1R-DT(XbaI&PstI)1.7 µL2.6 µL

Transformation

Nakamoto Tatsui

NameSampleCompetent CellsTotalPlate
9/9 Fusion1 antisense(pSB2C3)2µL20µL22µLCP
9/9 Fusion6 antisense(pSB2C3)2µL20µL22µLCP
9/9 aptamer 12_P(pSB2C3)2µL20µL22µLCP
9/9 aptamer 12_1&micro(pSB2C3)2µL20µL22µLCP
9/9 Fusion2 attenuator(pSB2C3) 2µL20µL22µLCP
9/11 Pcon-spinach-DT(pSB4K5)2µL20µL22µLK
9/11 RBS-lacZα-DT(pSB4K5)2µL20µL22µLK
9/11 Pcon-RBS-lacZα-DT(pSB4K5)2µL20µL22µLK
Plux+RBS-lysis1-DT2µL20µL22µLCP
Plux+RBS-lysis3-DT2µL20µL22µLCP
Pcon-pT181attenuator+RBS-lacZα-DT2µL20µL22µLAmp
Pcon-iuxR+Plux-GFP2µL20µL22µLAmp
Pcon-pT181attenuator+apt12_1R-DT2µL20µL22µLAmp
Plac(BBa_R0011)2µL20µL22µLAmp

Electrophoresis

Tatsui

LaneSample
1100bp ladder
2Pcon+RBS luxI-DT2
3Pcon+RBS luxI-DT3
4Pcon+RBS luxI-DT4
5Pcon+RBS luxI-DT5
6Pcon+RBS luxI-DT6
7Pcon+apF12_1R-DT1
8Pcon+apF12_1R-DT2
9Pcon+apF12_1R-DT3
10Pcon+apF12_1R-DT4
11DT+Pcon-pT181attenuator1
12F1attenuator(pSB1C3)
13100bp ladder

Igku 9119.jpg

Restriction Enzyme Digestion

Nakamoto

DNAEcoRIXbaISpeIPstIbufferBSAMilliQTotal
pSB4K5(EcoRI+SpeI)9.4µL 1µL 0µL 1µL 0µL 3µL 3µL 12.6µL30 µL
pSB4K5(EcoRI+SpeI) negative0.5µL 0µL 0µL 0µL 0µL 1µL 1µL 7.5µL 10µL
RBS-lacZα-DT(EcoRI+SpeI) 10µL 1µL 0µL 1µL 0µL 3µL 3µL 12µL 30µL
RBS-lacZα-DT(EcoRI+SpeI) negative 0.5µL 0µL 0µL 0µL 0µL 1µL 1µL 7.5µL 10µL
Ptet-pT181 antisense(SteI+PstI) 10.8µL 0µL 0µL 1µL 1µL 3µL 3µL 12µL 30µL
Ptet-pT181 antisense(SteI+PstI) negative 0.5µL 0µL 0µL 0µL 0µL 1µL 1µL 7.5µL 10µL
Pcon-RBS-lacZα-DT(EcoRI+SpeI) 7.1µL 1µL 0µL 1µL 0µL 3µL 3µL 14.9µL 30µL
Pcon-RBS-lacZα-DT(EcoRI+SpeI) negative 0.4µL 0µL 0µL 0µL 0µL 1µL 1µL 7.6µL 10µL
RBS-lysis2-DT(XbaI+PstI) 22µL 0µL 1µL 0µL 1µL 3µL 3µL 0µL 30µL
RBS-lysis2-DT(XbaI+PstI) negative 1.4µL 0µL 0µL 0µL 0µL 1µL 1µL 6.6µL 10µL

Liquid Culture

Nakamoto

Samplemedium
8/16 Master4 Plax
8/25 Pλ-laxI-2 　

PCR

Hirano

8/29 Pλ-RBS-luxI-DT(76.5ng/µL)10x buffer for KOD plus-ver.2KOD-plusdNTPMgSO4F primer(RBS-luxI-DT-cloning-fwd)R primer(RBS-luxI-DT-cloning-rev)MilliQtotal
0.32.50.52.51.50.750.7516.225
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　65°C　68°C--
5min30sec30sec1min30
8/31 Pbad/araC-RBS-RFP(1)(67.6ng/µL)10x buffer for KOD plus-ver.2KOD-plusdNTPMgSO4F primer(Pbad/araC(pSBlc3)-cloning-fwd)R primer(Pbad/araC(pSBlc3)-cloning-rev)MilliQtotal
0.32.50.52.51.50.750.7516.225
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　54°C　68°C--
5min30sec30sec3min 20sec30

Liquid Culture

Hirano

Samplemedium
9/4 RBS-lysis1+DT 3plusgrow(+CP) 3ml 37°C
9/4 RBS-lysis2+DT 1plusgrow(+CP) 3ml 37°C
9/4 RBS-lysis3+DT 3plusgrow(+CP) 3ml 37°C
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