Template:Kyoto/Notebook/Sep 8

From 2013.igem.org

(Difference between revisions)
(→Restriction Enzyme Digestion)
(→Ligation)
 
(15 intermediate revisions not shown)
Line 126: Line 126:
!state||colspan="2"|Vector(µL)||colspan="2"|Inserter(µL)||Ligation High ver.2
!state||colspan="2"|Vector(µL)||colspan="2"|Inserter(µL)||Ligation High ver.2
|-
|-
-
|experiment||9/8 Plac SpeI+PstI||4.3||9/3 pT181 attenuator||3.1||
+
|experiment||9/8 Plac SpeI+PstI||4.3||9/3 pT181 attenuator||3.1||3.5
|-
|-
-
|experiment||9/8 Plac SpeI+PstI||4.3||9/7 aptamer12-1R-DT XbaI+PstI||2.1||
+
|experiment||9/8 Plac SpeI+PstI||4.3||9/7 aptamer12-1R-DT XbaI+PstI||2.1||3.2
|-
|-
-
|experiment||9/8 Plac SpeI+PstI||4.3||9/8 spinach-DT XbaI+PstI||4.6||
+
|experiment||9/8 Plac SpeI+PstI||4.3||9/8 spinach-DT XbaI+PstI||4.6||3.5
|-
|-
-
|experiment||9/6 Plac SpeI+PstI||2||9/7 RBS-lysis2-DT XbaI+PstI||10||
+
|experiment||9/6 Plac SpeI+PstI||2||9/7 RBS-lysis2-DT XbaI+PstI||10||3.5
|-
|-
-
|experiment||9/8 Plac SpeI+PstI||4.3||9/7 RBS-lysis2-DT XbaI+PstI||10||
+
|experiment||9/8 Plac SpeI+PstI||4.3||9/7 RBS-lysis2-DT XbaI+PstI||10||3.5
|-
|-
-
|experiment||9/8 Ptet-pT181 anti||1.6||9/8 spinach-DT XbaI+PstI||4.3||
+
|experiment||9/8 Ptet-pT181 anti||1.6||9/8 spinach-DT XbaI+PstI||4.3||3.0
|-
|-
-
|experiment||9/6 Pcon SpeI+PstI||1.4||9/8 RBS-laczα-DT XbaI+PstI||4.8||
+
|experiment||9/6 Pcon SpeI+PstI||1.4||9/8 RBS-laczα-DT XbaI+PstI||4.8||3.1
|-
|-
-
|experiment||9/8 Pcon-pT181 attenuator SpeI+PstI||3.4||9/7 aptamer12-1R-DT XbaI+PstI||1.9||
+
|experiment||9/8 Pcon-pT181 attenuator SpeI+PstI||3.4||9/7 aptamer12-1R-DT XbaI+PstI||1.9||2.7
|-
|-
-
|experiment||9/6 Pcon SpeI+PstI||1.4||9/7 aptamer12-1R-DT XbaI+PstI||2.1||
+
|experiment||9/6 Pcon SpeI+PstI||1.4||9/7 aptamer12-1R-DT XbaI+PstI||2.1||1.3
|-
|-
-
|experiment||9/8 DT||3.0||9/8 Pcon-pT181 attenuator EcoRI+SpeI||7.9||
+
|experiment||9/8 DT||3.0||9/8 Pcon-pT181 attenuator EcoRI+SpeI||7.9||3.5
|-
|-
-
|experiment||9/6 Plux SpeI+PstI||2||9/7 RBS-lysis1-DT XbaI+PstI||7.2||
+
|experiment||9/6 Plux SpeI+PstI||2||9/7 RBS-lysis1-DT XbaI+PstI||7.2||3.5
|-
|-
-
|experiment||9/8 Plac SpeI+PstI||4.3||9/7 RBS-lysis1-DT XbaI+PstI||7.2||
+
|experiment||9/8 Plac SpeI+PstI||4.3||9/7 RBS-lysis1-DT XbaI+PstI||7.2||3.5
|-
|-
-
|experiment||9/4 Plac SpeI+PstI||1.7||9/8 RBS-laczα-DT XbaI+PstI||4.8||
+
|experiment||9/4 Plac SpeI+PstI||1.7||9/8 RBS-laczα-DT XbaI+PstI||4.8||3.3
|}
|}
</div>
</div>
Line 165: Line 165:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||9/8 Pcon-spin-DT||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
+
! ||9/8 Pcon-spinach-DT||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts||7.9&micro;L|| 1&micro;L|| 1&micro;L|| 0&micro;L|| 0&micro;L|| 3&micro;L|| 3&micro;L||14.1&micro;L|| 30&micro;L
|2 cuts||7.9&micro;L|| 1&micro;L|| 1&micro;L|| 0&micro;L|| 0&micro;L|| 3&micro;L|| 3&micro;L||14.1&micro;L|| 30&micro;L
Line 172: Line 172:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||8/21 F1 atte||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
+
! ||8/21 F1 attenuator||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts|| 6.5&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L||15.5&micro;L||30&micro;L
|2 cuts|| 6.5&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L||15.5&micro;L||30&micro;L
Line 179: Line 179:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||8/21 F3m2 atte||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
+
! ||8/21 F3m2 attenuator||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts||6.0&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||16&micro;L||30&micro;L
|2 cuts||6.0&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||16&micro;L||30&micro;L
Line 214: Line 214:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||9/4 spin-DT||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
+
! ||9/4 spinach-DT||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
||2 cuts||5.4&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||16.6&micro;L||30&micro;L
||2 cuts||5.4&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||16.6&micro;L||30&micro;L
Line 242: Line 242:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||8/20 J23100 RBS-lacZα-DT-(1)||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
+
! ||8/20 J23100 RBS-lacZ&alpha;-DT-(1)||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts|||7&micro;L||0.5&micro;L||0.5&micro;L||0&micro;L||0&micro;L||3&micro;L||3&micro;L||16&micro;L||30&micro;L
|2 cuts|||7&micro;L||0.5&micro;L||0.5&micro;L||0&micro;L||0&micro;L||3&micro;L||3&micro;L||16&micro;L||30&micro;L
Line 249: Line 249:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||8/20 RBS-lacZα-DT||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
+
! ||8/20 RBS-lacZ&alpha;-DT||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts||10&micro;L||0.5&micro;L||0.5&micro;L||0&micro;L||0&micro;L||3&micro;L||3&micro;L||13&micro;L||30&micro;L
|2 cuts||10&micro;L||0.5&micro;L||0.5&micro;L||0&micro;L||0&micro;L||3&micro;L||3&micro;L||13&micro;L||30&micro;L
Line 258: Line 258:
===Transformation===
===Transformation===
-
<!-- こっから -->
 
<div class="experiment">
<div class="experiment">
-
<span class="author">じっけんしゃ</span>
+
<span class="author">Nakamoto & Ashida</span>
{| class="wikitable"
{| class="wikitable"
!Name||Sample(&micro;L)||Competent Cells(&micro;L)||Total(&micro;L)||Plate
!Name||Sample(&micro;L)||Competent Cells(&micro;L)||Total(&micro;L)||Plate
Line 266: Line 265:
|9/8 Pcont+aptamer 12-1R-DT||1||10||11||Amp
|9/8 Pcont+aptamer 12-1R-DT||1||10||11||Amp
|-
|-
-
|9/8 Pcon+RBS-lacZα-DT||1||10||11||Amp
+
|9/8 Pcon+RBS-lacZ&alpha;-DT||1||10||11||Amp
|-
|-
|9/8 Pcon-pT181 attenuator+aptamer 12-1R-DT||1||10||11||Amp
|9/8 Pcon-pT181 attenuator+aptamer 12-1R-DT||1||10||11||Amp
Line 284: Line 283:
|9/8 Plux+RBS-lysis2-DT||1||10||11||CP
|9/8 Plux+RBS-lysis2-DT||1||10||11||CP
|-
|-
-
|9/8 Ptet+RBS-lacZα-DT||1||10||11||CP
+
|9/8 Ptet+RBS-lacZ&alpha;-DT||1||10||11||CP
|-
|-
|9/8 Ptet-pT181 anti+spinach-DT||1||10||11||CP
|9/8 Ptet-pT181 anti+spinach-DT||1||10||11||CP
Line 290: Line 289:
|9/8 Pcon-pT181 attenuator+DT||1||10||11||CP
|9/8 Pcon-pT181 attenuator+DT||1||10||11||CP
|}
|}
 +
</div>
===Electrophoresis===
===Electrophoresis===
-
 
-
<!-- こっから -->
 
<div class="experiment">
<div class="experiment">
-
<span class="author">じっけんしゃ</span>
+
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
!Lane||Sample||Enzyme1||Enzyme2
!Lane||Sample||Enzyme1||Enzyme2
Line 301: Line 299:
|1||100bp ladder|| ||  
|1||100bp ladder|| ||  
|-
|-
-
|2||Pcon-spin-DT||EcoRI||SpeI
+
|2||Pcon-spinach-DT||EcoRI||SpeI
|-
|-
-
|3||Pcon-spin-DT|| ||  
+
|3||Pcon-spinach-DT|| ||  
|-
|-
|4||Fusion1 attenuator||XbaI||PstI
|4||Fusion1 attenuator||XbaI||PstI
Line 338: Line 336:
|1||spinach-DT|| ||  
|1||spinach-DT|| ||  
|-
|-
-
|2||J23100-RBS-lacZα-DT||EcoRI||SpeI
+
|2||J23100-RBS-lacZ&alpha;-DT||EcoRI||SpeI
|-
|-
-
|3||J23100-RBS-lacZα-DT|| ||  
+
|3||J23100-RBS-lacZ&alpha;-DT|| ||  
|-
|-
|4||100bp ladder|| ||  
|4||100bp ladder|| ||  
|-
|-
-
|5||RBS-lacZα-DT||EcoRI||SpeI
+
|5||RBS-lacZ&alpha;-DT||EcoRI||SpeI
|-
|-
-
|6||RBS-lacZα-DT|| ||  
+
|6||RBS-lacZ&alpha;-DT|| ||  
|-
|-
|7||RBS-lysis1-DT||XbaI||PstI
|7||RBS-lysis1-DT||XbaI||PstI
Line 372: Line 370:
|}
|}
[[File:Igku_986.png]]
[[File:Igku_986.png]]
 +
</div>
===Gel Extraction===
===Gel Extraction===
-
<!-- こっから -->
 
<div class="experiment">
<div class="experiment">
-
<span class="author">じっけんしゃ</span>
+
<span class="author">Ashida and Nakamoto</span>
{| class="wikitable"
{| class="wikitable"
!Lane||DNA||Enzyme
!Lane||DNA||Enzyme
Line 382: Line 380:
|1||100bp ladder||  
|1||100bp ladder||  
|-
|-
-
|2||Pcon-spin-DT||EcoRI+SpeI
+
|2||Pcon-spinach-DT||EcoRI+SpeI
|-
|-
-
|3||Pcon-Spin-DT||EcoRI+SpeI
+
|3||Pcon-Spinach-DT||EcoRI+SpeI
|-
|-
|5||Fusion 1 attenuator||XbaI+PstI
|5||Fusion 1 attenuator||XbaI+PstI
Line 394: Line 392:
|9||Fusion3m2 attenuator||XbaI+PstI
|9||Fusion3m2 attenuator||XbaI+PstI
|-
|-
-
|11||Fusion 1 anti||XbaI+PstI
+
|11||Fusion 1 antisense||XbaI+PstI
|-
|-
-
|12||Fusion 1 anti||XbaI+PstI
+
|12||Fusion 1 antisense||XbaI+PstI
|-
|-
|}
|}
Line 403: Line 401:
!Name||quantity||concentration[&micro;g/mL]||260/280||260/230
!Name||quantity||concentration[&micro;g/mL]||260/280||260/230
|-
|-
-
|Pcon-spin-DT(EcoRI+SpeI)|| ||10.5||1.86||0.39
+
|Pcon-spinach-DT(EcoRI+SpeI)|| ||10.5||1.86||0.39
|-
|-
|Fusion 1 attenuator(XbaI+PstI)|| ||7.0||1.71||0.27
|Fusion 1 attenuator(XbaI+PstI)|| ||7.0||1.71||0.27
Line 409: Line 407:
|Fusion3m2 attenuator(XbaI+PstI)|| ||8.6||1.75||0.47
|Fusion3m2 attenuator(XbaI+PstI)|| ||8.6||1.75||0.47
|-
|-
-
|Fusion 1 anti(XbaI+PstI)|| ||7.6||1.87||0.43
+
|Fusion 1 antisense(XbaI+PstI)|| ||7.6||1.87||0.43
|-
|-
|}
|}
Line 417: Line 415:
|1||100bp ladder||  
|1||100bp ladder||  
|-
|-
-
|2||Fusion6 anti||XbaI+PstI
+
|2||Fusion6 antisense||XbaI+PstI
|-
|-
-
|3||Fusion6 anti||XbaI+PstI
+
|3||Fusion6 antisense||XbaI+PstI
|-
|-
|5||aptamer12_1M||XbaI+PstI
|5||aptamer12_1M||XbaI+PstI
Line 431: Line 429:
!Name||quantity||concentration[&micro;g/mL]||260/280||260/230
!Name||quantity||concentration[&micro;g/mL]||260/280||260/230
|-
|-
-
|Fusion6 anti(XbaI+PstI)|| ||5.0||1.76||0.20
+
|Fusion6 antisense(XbaI+PstI)|| ||5.0||1.76||0.20
|-
|-
|aptamer12_1M(XbaI+PstI)|| ||3.3||2.31||0.04
|aptamer12_1M(XbaI+PstI)|| ||3.3||2.31||0.04
Line 465: Line 463:
|1||100bp ladder||  
|1||100bp ladder||  
|-
|-
-
|2||J23100-RBS-laczα-DT||EcoRI+SpeI
+
|2||J23100-RBS-lacz&alpha;-DT||EcoRI+SpeI
|-
|-
-
|3||J23100-RBS-laczα-DT||EcoRI+SpeI
+
|3||J23100-RBS-lacz&alpha;-DT||EcoRI+SpeI
|-
|-
-
|5||RBS-laczα-DT||EcoRI+SpeI
+
|5||RBS-lacz&alpha;-DT||EcoRI+SpeI
|-
|-
-
|6||RBS-laczα-DT||EcoRI+SpeI
+
|6||RBS-lacz&alpha;-DT||EcoRI+SpeI
|-
|-
|8||RBS-lysis1-DT||XbaI+PstI
|8||RBS-lysis1-DT||XbaI+PstI
Line 487: Line 485:
!Name||quantity||concentration[&micro;g/mL]||260/280||260/230
!Name||quantity||concentration[&micro;g/mL]||260/280||260/230
|-
|-
-
|Pcon-RBS-laczα-DT(EcoRI+SpeI)|| ||5.6||2.03||0.25
+
|Pcon-RBS-lacz&alpha;-DT(EcoRI+SpeI)|| ||5.6||2.03||0.25
|-
|-
-
|RBS-laczα-DT(EcoRI+SpeI)|| ||4.4||2.04||0.33
+
|RBS-lacz&alpha;-DT(EcoRI+SpeI)|| ||4.4||2.04||0.33
|-
|-
|RBS-lysis1-DT(XbaI+PstI)|| ||5.9||2.22||0.53
|RBS-lysis1-DT(XbaI+PstI)|| ||5.9||2.22||0.53
Line 505: Line 503:
|3||RBS-lysis3-DT||XbaI+PstI
|3||RBS-lysis3-DT||XbaI+PstI
|-
|-
-
|5||Pcon-pT181 anti||SpeI+PstI
+
|5||Pcon-pT181 antisense||SpeI+PstI
|-
|-
-
|6||Pcon-pT181 anti||SpeI+PstI
+
|6||Pcon-pT181 antisense||SpeI+PstI
|-
|-
|}
|}
Line 517: Line 515:
|RBS-lysis3-DT(XbaI+PstI)|| ||23.4||1.72||0.88
|RBS-lysis3-DT(XbaI+PstI)|| ||23.4||1.72||0.88
|-
|-
-
|Pcon-pT181 anti(SpeI+PstI)|| ||31.3||1.86||0.92
+
|Pcon-pT181 antisense(SpeI+PstI)|| ||31.3||1.86||0.92
|-
|-
|}
|}
 +
</div>
===Ligation===
===Ligation===
-
 
-
<!-- こっから -->
 
<div class="experiment">
<div class="experiment">
-
<span class="author">じっけんしゃ</span>
+
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
-
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
+
!state||colspan="2"|Vector||colspan="2"|Inserter
|-
|-
-
|experiment||Pcon-spin-DT(EcoRI&SpeI)||10.5&micro;g/mL||pSB6A1(EcoRI&SpeI)||8.9&micro;g/mL
+
|experiment||Pcon-spinach-DT(EcoRI&SpeI)||10.5&micro;g/mL||pSB6A1(EcoRI&SpeI)||8.9&micro;g/mL
|-
|-
-
|experiment||Pcon-spin-DT(EcoRI&SpeI)||10.5&micro;g/mL||pSB4K5(EcoRI&SpeI)||21.8&micro;g/mL
+
|experiment||Pcon-spinach-DT(EcoRI&SpeI)||10.5&micro;g/mL||pSB4K5(EcoRI&SpeI)||21.8&micro;g/mL
|-
|-
-
|experiment||Pcon-pT181 anti(SpeI+PstI)||31.3&micro;g/mL||spin-DT(XbaI+PstI)||10.1&micro;g/mL
+
|experiment||Pcon-pT181 anti(SpeI+PstI)||31.3&micro;g/mL||spinach-DT(XbaI+PstI)||10.1&micro;g/mL
|-
|-
|experiment||Plac(SpeI+PstI)||11.6&micro;g/mL||RBS-lysis3-DT||23.4&micro;g/mL
|experiment||Plac(SpeI+PstI)||11.6&micro;g/mL||RBS-lysis3-DT||23.4&micro;g/mL
Line 539: Line 536:
|experiment||Plux(SpeI+PstI)||25&micro;g/mL||RBS-lysis3-DT||23.4&micro;g/mL
|experiment||Plux(SpeI+PstI)||25&micro;g/mL||RBS-lysis3-DT||23.4&micro;g/mL
|-
|-
-
|experiment||Ptet-pT181 anti(SpeI+PstI)||30.6&micro;g/mL||spin-DT(XbaI+PstI)||10.1&micro;g/mL
+
|experiment||Ptet-pT181 anti(SpeI+PstI)||30.6&micro;g/mL||spinach-DT(XbaI+PstI)||10.1&micro;g/mL
|-
|-
-
|experiment||Pcon-pT181 attenuator(SpeI+PstI)||14.5&micro;g/mL||RBS-laczα-DT(XbaI+PstI)||4.4&micro;g/mL
+
|experiment||Pcon-pT181 attenuator(SpeI+PstI)||14.5&micro;g/mL||RBS-lacz&alpha;-DT(XbaI+PstI)||4.4&micro;g/mL
|-
|-
|}
|}
 +
</div>
===PCR===
===PCR===
Line 623: Line 621:
===Gel Extraction===
===Gel Extraction===
-
<!-- こっから -->
 
<div class="experiment">
<div class="experiment">
-
<span class="author">じっけんしゃ</span>
+
<span class="author">Yoshida</span>
{| class="wikitable"
{| class="wikitable"
-
!Lane||DNA||Enzyme
+
!Lane||DNA
|-
|-
|1||100bp ladder
|1||100bp ladder
Line 652: Line 649:
|-
|-
|}
|}
-
 
+
[[File:9898.jpg]]<br>
 +
[[File:9897.jpg]]<br>
{| class="wikitable"
{| class="wikitable"
-
!Lane||DNA||Enzyme
+
!Lane||DNA
-
|-
+
-
|1||100bp ladder
+
-
|-
+
-
|2||KaiA
+
-
|-
+
-
|3||KaiA
+
-
|-
+
-
|4||KaiB
+
-
|-
+
-
|5||KaiB
+
-
|-
+
-
|6||DT
+
-
|-
+
-
|7||DT
+
-
|-
+
-
|8||P-KaiBC
+
-
|-
+
-
|9||P-KaiBC
+
-
|-
+
-
|10||nega
+
-
|-
+
-
|11||100bp ladder
+
-
|-
+
-
|}
+
-
 
+
-
{| class="wikitable"
+
-
!Lane||DNA||Enzyme
+
|-
|-
|1||Plac
|1||Plac
Line 696: Line 667:
|-
|-
|}
|}
 +
[[File:9899.jpg]]<br>
 +
[[File:991.jpg]]<br>
 +
</div>

Latest revision as of 14:25, 27 September 2013

Contents

Sep 8

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder
2RBS-lysis1-DTXbaI & PstI
3RBS-lysis1-DTXbaI & PstI
5RBS-lysis3-DTXbaI & PstI
6RBS-lysis3-DTXbaI & PstI

Igku 981b.png
Igku 981a.png

Nameconcentration[µg/mL]260/280260/230
RBS-lysis1-DT (XbaI & PstI)5.31.980.06
RBS-lysis3-DT (XbaI & PstI)5.31.980.06
LaneDNAEnzyme
1100bp ladder
3Pcon-pT181 attenuatorEcoRI & SpeI
4Pcon-pT181 attenuatorEcoRI & SpeI
6Spinach-DTXbaI & PstI
7Spinach-DTXbaI & PstI
9RBS-lacZα-DTXbaI & PstI
10RBS-lacZα-DTXbaI & PstI

Igku 982b.png
Igku 982a.png

Nameconcentration[µg/mL]260/280260/230
Pcon-pT181 attenuator (EcoRI & SpeI)5.21.820.36
Spinach-DT (XbaI & PstI)8.01.870.25
RBS-lacZα-DT (XbaI & PstI)10.31.710.54
LaneDNAEnzyme
11kbp ladder
2Ptet-pT181 antisenseEcoRI & PstI
3Ptet-pT181 antisenseEcoRI & PstI
5pSB4K5EcoRI & SpeI
6pSB4K5EcoRI & SpeI

Igku 983b.png
Igku 983a.png

Nameconcentration[µg/mL]260/280260/230
Ptet-pT181 antisense(EcoRI & PstI)30.61.841.16
pSB4K5 (EcoRI & SpeI)21.81.870.98
LaneDNAEnzyme
11kbp ladder
2Pcon-pT181 attenuatorSpeI & PstI
3Pcon-pT181 attenuatorSpeI & PstI
5PlacSpeI & PstI
6PlacSpeI & PstI
8pSB1C3EcoRI & SpeI
9pSB1C3EcoRI & SpeI
11pSB1C3XbaI & PstI
12pSB1C3XbaI & PstI

Igku 984b.png
Igku 984a.png

Nameconcentration[µg/mL]260/280260/230
Pcon-pT181 attenuator (SpeI & PstI)14.51.911.09
Plac (SpeI & PstI)11.61.741.19
pSB1C3 (EcoRI & SpeI)5.71.740.34
pSB1C3 (XbaI & PstI)5.62.000.37

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
Pcon-pT181 anti243.01.901.81
Pcon-Spinach-DT253.71.571.51

Ligation

Kojima

stateVector(µL)Inserter(µL)Ligation High ver.2
experiment9/8 Plac SpeI+PstI4.39/3 pT181 attenuator3.13.5
experiment9/8 Plac SpeI+PstI4.39/7 aptamer12-1R-DT XbaI+PstI2.13.2
experiment9/8 Plac SpeI+PstI4.39/8 spinach-DT XbaI+PstI4.63.5
experiment9/6 Plac SpeI+PstI29/7 RBS-lysis2-DT XbaI+PstI103.5
experiment9/8 Plac SpeI+PstI4.39/7 RBS-lysis2-DT XbaI+PstI103.5
experiment9/8 Ptet-pT181 anti1.69/8 spinach-DT XbaI+PstI4.33.0
experiment9/6 Pcon SpeI+PstI1.49/8 RBS-laczα-DT XbaI+PstI4.83.1
experiment9/8 Pcon-pT181 attenuator SpeI+PstI3.49/7 aptamer12-1R-DT XbaI+PstI1.92.7
experiment9/6 Pcon SpeI+PstI1.49/7 aptamer12-1R-DT XbaI+PstI2.11.3
experiment9/8 DT3.09/8 Pcon-pT181 attenuator EcoRI+SpeI7.93.5
experiment9/6 Plux SpeI+PstI29/7 RBS-lysis1-DT XbaI+PstI7.23.5
experiment9/8 Plac SpeI+PstI4.39/7 RBS-lysis1-DT XbaI+PstI7.23.5
experiment9/4 Plac SpeI+PstI1.79/8 RBS-laczα-DT XbaI+PstI4.83.3

Restriction Enzyme Digestion

Kojima

9/8 Pcon-pT181 antiEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts8.2µL 0µL 1µL 0µL 1µL 3µL 3µL 13.8µL 30µL
NC 0.4µL 0µL 0µL 0µL 0µL 1µL 1µL 7.6µL 10µL
9/8 Pcon-spinach-DTEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts7.9µL 1µL 1µL 0µL 0µL 3µL 3µL14.1µL 30µL
NC 0.4µL 0µL 0µL 0µL 0µL 1µL 1µL7.6µL 10µL
8/21 F1 attenuatorEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts 6.5µL 0µL 0µL 1µL 1µL 3µL 3µL15.5µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/21 F3m2 attenuatorEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts6.0µL0µL0µL1µL1µL3µL3µL16µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/21 F1 antiEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts6.7µL0µL0µL1µL1µL3µL3µL15.3µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/21 F6 antiEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts6.0µL0µL0µL1µL1µL3µL3µL16µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/21 tetR aptamer 12_1MEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts5.3µL1µL1µL0µL0µL3µL3µL16.7µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/21 tetR aptamer 12_PEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts13.3µL1µL1µL0µL0µL3µL3µL8.7µL30µL
NC0.7µL0µL0µL0µL0µL1µL1µL7.3µL10µL
9/4 spinach-DTEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts5.4µL0µL0µL1µL1µL3µL3µL16.6µL30µL
NC0.3µL0µL0µL0µL0µL1µL1µL7.7µL10µL
9/7 RBS-lysis1-DTEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts10µL0µL0µL1µL1µL3µL3µL12µL30µL
NC0.5µL0µL0µL0µL0µL1µL1µL7.5µL10µL
9/6 RBS-lysis2-DTEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts13µL0µL0µL1µL1µL3µL3µL9µL30µL
NC0.7µL0µL0µL0µL0µL1µL1µL7.3µL10µL
9/7 RBS-lysis3-DTEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts10µL0µL0µL1µL1µL3µL3µL12µL30µL
NC0.5µL0µL0µL0µL0µL1µL1µL7.5µL10µL
8/20 J23100 RBS-lacZα-DT-(1)EcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts7µL0.5µL0.5µL0µL0µL3µL3µL16µL30µL
NC0.5µL0µL0µL0µL0µL1µL1µL7.5µL10µL
8/20 RBS-lacZα-DTEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts10µL0.5µL0.5µL0µL0µL3µL3µL13µL30µL
NC0.5µL0µL0µL0µL0µL1µL1µL7.5µL10µL

Transformation

Nakamoto & Ashida

NameSample(µL)Competent Cells(µL)Total(µL)Plate
9/8 Pcont+aptamer 12-1R-DT11011Amp
9/8 Pcon+RBS-lacZα-DT11011Amp
9/8 Pcon-pT181 attenuator+aptamer 12-1R-DT11011Amp
9/8 Plac+RBS-lysis1-DT22022CP
9/8 Plac+RBS-lysis2-DT22022CP
9/8 Plac+aptamer 12-1R-DT22022CP
9/8 Plac+spinach22022CP
9/8 Plac+pT181 attenuator22022CP
9/8 Plux+RBS-lysis1-DT11011CP
9/8 Plux+RBS-lysis2-DT11011CP
9/8 Ptet+RBS-lacZα-DT11011CP
9/8 Ptet-pT181 anti+spinach-DT11011CP
9/8 Pcon-pT181 attenuator+DT11011CP

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder
2Pcon-spinach-DTEcoRISpeI
3Pcon-spinach-DT
4Fusion1 attenuatorXbaIPstI
5Fusion1 attenuator
6Fusion3m2 attenuatorXbaIPstI
7Fusion3m2 attenuator
8Fusion1 antiXbaIPstI
9Fusion1 anti
10Fusion6 antiXbaIPstI
11Fusion6 anti
12aptamer 12-1MXbaIPstI
13aptamer 12-1M
14aptamer 12-PXbaIPstI
15aptamer 12-P
16spinach-DTXbaIPstI

Igku 985.png

LaneSampleEnzyme1Enzyme2
1spinach-DT
2J23100-RBS-lacZα-DTEcoRISpeI
3J23100-RBS-lacZα-DT
4100bp ladder
5RBS-lacZα-DTEcoRISpeI
6RBS-lacZα-DT
7RBS-lysis1-DTXbaIPstI
8RBS-lysis1-DT
LaneSampleEnzyme1Enzyme2
1100bp ladder
2RBS-lysis2-DTXbaIPstI
3RBS-lysis2-DT
4RBS-lysis3-DTXbaIPstI
5RBS-lysis3-DT
6Pcon pT181 antiSpeIPstI
7Pcon pT181 anti

Igku 986.png

Gel Extraction

Ashida and Nakamoto

LaneDNAEnzyme
1100bp ladder
2Pcon-spinach-DTEcoRI+SpeI
3Pcon-Spinach-DTEcoRI+SpeI
5Fusion 1 attenuatorXbaI+PstI
6Fusion 1 attenuatorXbaI+PstI
8Fusion3m2 attenuatorXbaI+PstI
9Fusion3m2 attenuatorXbaI+PstI
11Fusion 1 antisenseXbaI+PstI
12Fusion 1 antisenseXbaI+PstI

Igku 987.png

Namequantityconcentration[µg/mL]260/280260/230
Pcon-spinach-DT(EcoRI+SpeI) 10.51.860.39
Fusion 1 attenuator(XbaI+PstI) 7.01.710.27
Fusion3m2 attenuator(XbaI+PstI) 8.61.750.47
Fusion 1 antisense(XbaI+PstI) 7.61.870.43
LaneDNAEnzyme
1100bp ladder
2Fusion6 antisenseXbaI+PstI
3Fusion6 antisenseXbaI+PstI
5aptamer12_1MXbaI+PstI
6aptamer12_1MXbaI+PstI

Igku 988b.png
Igku 988a.png

Namequantityconcentration[µg/mL]260/280260/230
Fusion6 antisense(XbaI+PstI) 5.01.760.20
aptamer12_1M(XbaI+PstI) 3.32.310.04
LaneDNAEnzyme
1100bp ladder
2aptamer12_PXbaI+PstI
3aptamer12_PXbaI+PstI
5spinach-DTEcoRI+SpeI
6spinach-DTEcoRI+SpeI

Igku 989b.png
Igku 989a.png

Namequantityconcentration[µg/mL]260/280260/230
aptamer12_P(XbaI+PstI) 3.41.720.26
spinach-DT(EcoRI+SpeI) 10.11.570.47
LaneDNAEnzyme
1100bp ladder
2J23100-RBS-laczα-DTEcoRI+SpeI
3J23100-RBS-laczα-DTEcoRI+SpeI
5RBS-laczα-DTEcoRI+SpeI
6RBS-laczα-DTEcoRI+SpeI
8RBS-lysis1-DTXbaI+PstI
9RBS-lysis1-DTXbaI+PstI
11RBS-lysis2-DTXbaI+PstI
12RBS-lysis2-DTXbaI+PstI

Igku 9810b.png
Igku 9810a.png

Namequantityconcentration[µg/mL]260/280260/230
Pcon-RBS-laczα-DT(EcoRI+SpeI) 5.62.030.25
RBS-laczα-DT(EcoRI+SpeI) 4.42.040.33
RBS-lysis1-DT(XbaI+PstI) 5.92.220.53
RBS-lysis2-DT(XbaI+PstI) 5.11.670.48
LaneDNAEnzyme
1100bp ladder
2RBS-lysis3-DTXbaI+PstI
3RBS-lysis3-DTXbaI+PstI
5Pcon-pT181 antisenseSpeI+PstI
6Pcon-pT181 antisenseSpeI+PstI

Igku 9811b.png
Igku 9811a.png

Namequantityconcentration[µg/mL]260/280260/230
RBS-lysis3-DT(XbaI+PstI) 23.41.720.88
Pcon-pT181 antisense(SpeI+PstI) 31.31.860.92

Ligation

No name

stateVectorInserter
experimentPcon-spinach-DT(EcoRI&SpeI)10.5µg/mLpSB6A1(EcoRI&SpeI)8.9µg/mL
experimentPcon-spinach-DT(EcoRI&SpeI)10.5µg/mLpSB4K5(EcoRI&SpeI)21.8µg/mL
experimentPcon-pT181 anti(SpeI+PstI)31.3µg/mLspinach-DT(XbaI+PstI)10.1µg/mL
experimentPlac(SpeI+PstI)11.6µg/mLRBS-lysis3-DT23.4µg/mL
experimentPlux(SpeI+PstI)25µg/mLRBS-lysis3-DT23.4µg/mL
experimentPtet-pT181 anti(SpeI+PstI)30.6µg/mLspinach-DT(XbaI+PstI)10.1µg/mL
experimentPcon-pT181 attenuator(SpeI+PstI)14.5µg/mLRBS-laczα-DT(XbaI+PstI)4.4µg/mL

PCR

No name

KaiA(plasmid)KOD plus10x bufferdNTPMgSO4Primer(661-RBS-KaiA-fwd)Primer(kaiA-662-rev-ver2)MilliQtotal
10.52.52.51.50.750.7515.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s28s30cycles
KaiB(plasmid)KOD plus10x bufferdNTPMgSO4Primer(662-RBS-KaiB-fwd-ver2)Primer(kaiB-663-rev-ver2)MilliQtotal
10.52.52.51.50.750.7515.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s28s30cycles
DT(plasmid)KOD plus10x bufferdNTPMgSO4Primer(664-DT-fwd-ver2)Primer(DT-suffix-660-rev-ver2)MilliQtotal
0.10.52.52.51.50.750.7516.425
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s28s30cycles
P-KaiB(PCR prpduct)KOD plus10x bufferdNTPMgSO4Primer(prefix-PkaiBC-fwd-ver2)Primer(PkaiBC-suffix--662-rev-ver2)MilliQtotal
0.70.52.52.51.50.750.7515.825
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s28s30cycles
Plac(1)(plasmid)KOD plus10x bufferdNTPMgSO4Primer(GG0-pSBIC3-fwd-ver2)Primer(Plac-GG1-rev-ver2)MilliQtotal
0.130.52.52.51.50.750.7516.3725
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s60s30cycles
KaiC(plasmid)KOD plus10x bufferdNTPMgSO4Primer(GG3-RBS-KaiC-fwd-ver2)Primer(KaiC-GG4-rev-ver2)MilliQtotal
10.52.52.51.50.750.7515.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s60s30cycles

Gel Extraction

Yoshida

LaneDNA
1100bp ladder
2KaiA
3KaiA
4KaiB
5KaiB
6DT
7DT
8P-KaiBC
9P-KaiBC
10nega
11100bp ladder

9898.jpg
9897.jpg

LaneDNA
1Plac
2Plac
3KaiC
4KaiC
5nega
61kb ladder

9899.jpg
991.jpg

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