Template:Kyoto/Notebook/Sep 8

From 2013.igem.org

(Difference between revisions)
(→Gel Extraction)
(→Ligation)
 
(26 intermediate revisions not shown)
Line 16: Line 16:
|6||RBS-lysis3-DT||XbaI & PstI
|6||RBS-lysis3-DT||XbaI & PstI
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:igku_981b.png]]<br>
-
[[File:igku_xxafterxx.xxx]]<br>
+
[[File:igku_981a.png]]<br>
{| class="wikitable"
{| class="wikitable"
!Name||concentration[&micro;g/mL]||260/280||260/230
!Name||concentration[&micro;g/mL]||260/280||260/230
Line 42: Line 42:
|10||RBS-lacZ&alpha;-DT||XbaI & PstI
|10||RBS-lacZ&alpha;-DT||XbaI & PstI
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:igku_982b.png]]<br>
-
[[File:igku_xxafterxx.xxx]]<br>
+
[[File:igku_982a.png]]<br>
{| class="wikitable"
{| class="wikitable"
!Name||concentration[&micro;g/mL]||260/280||260/230
!Name||concentration[&micro;g/mL]||260/280||260/230
Line 66: Line 66:
|6||pSB4K5||EcoRI & SpeI
|6||pSB4K5||EcoRI & SpeI
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:igku_983b.png]]<br>
-
[[File:igku_xxafterxx.xxx]]<br>
+
[[File:igku_983a.png]]<br>
{| class="wikitable"
{| class="wikitable"
!Name||concentration[&micro;g/mL]||260/280||260/230
!Name||concentration[&micro;g/mL]||260/280||260/230
Line 96: Line 96:
|12||pSB1C3||XbaI & PstI
|12||pSB1C3||XbaI & PstI
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:igku_984b.png]]<br>
-
[[File:igku_xxafterxx.xxx]]<br>
+
[[File:igku_984a.png]]<br>
{| class="wikitable"
{| class="wikitable"
!Name||concentration[&micro;g/mL]||260/280||260/230
!Name||concentration[&micro;g/mL]||260/280||260/230
Line 120: Line 120:
|Pcon-Spinach-DT||253.7||1.57||1.51
|Pcon-Spinach-DT||253.7||1.57||1.51
|}
|}
 +
===Ligation===
 +
<div class="experiment">
 +
<span class="author">Kojima</span>
 +
{| class="wikitable"
 +
!state||colspan="2"|Vector(&micro;L)||colspan="2"|Inserter(&micro;L)||Ligation High ver.2
 +
|-
 +
|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||3.2
 +
|-
 +
|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||3.5
 +
|-
 +
|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||3.0
 +
|-
 +
|experiment||9/6 Pcon SpeI+PstI||1.4||9/8 RBS-lacz&alpha;-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||2.7
 +
|-
 +
|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||3.5
 +
|-
 +
|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||3.5
 +
|-
 +
|experiment||9/4 Plac SpeI+PstI||1.7||9/8 RBS-lacz&alpha;-DT XbaI+PstI||4.8||3.3
 +
|}
 +
</div>
===Restriction Enzyme Digestion===
===Restriction Enzyme Digestion===
-
<!-- こっから -->
 
<div class="experiment">
<div class="experiment">
-
<span class="author">じっけんしゃ</span>
+
<span class="author">Kojima</span>
{| class="wikitable"
{| class="wikitable"
-
! ||9/8 Pcon-pT181 anti||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||9/8 Pcon-pT181 anti||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts||8.2&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L|| 13.8&micro;L|| 30&micro;L
|2 cuts||8.2&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L|| 13.8&micro;L|| 30&micro;L
Line 133: Line 165:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||9/8 Pcon-spin-DT||E||S||X||P||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 140: Line 172:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||8/21 F1 atte||E||S||X||P||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 147: Line 179:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||8/21 F3m2 atte||E||S||X||P||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 154: Line 186:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||8/21 F1 anti||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||8/21 F1 anti||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts||6.7&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||15.3&micro;L||30&micro;L
|2 cuts||6.7&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||15.3&micro;L||30&micro;L
Line 161: Line 193:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||8/21 F6 anti||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||8/21 F6 anti||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 168: Line 200:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||8/21 tetR aptamer 12_1M||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||8/21 tetR aptamer 12_1M||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts||5.3&micro;L||1&micro;L||1&micro;L||0&micro;L||0&micro;L||3&micro;L||3&micro;L||16.7&micro;L||30&micro;L
|2 cuts||5.3&micro;L||1&micro;L||1&micro;L||0&micro;L||0&micro;L||3&micro;L||3&micro;L||16.7&micro;L||30&micro;L
Line 175: Line 207:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||8/21 tetR aptamer 12_P||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||8/21 tetR aptamer 12_P||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts||13.3&micro;L||1&micro;L||1&micro;L||0&micro;L||0&micro;L||3&micro;L||3&micro;L||8.7&micro;L||30&micro;L
|2 cuts||13.3&micro;L||1&micro;L||1&micro;L||0&micro;L||0&micro;L||3&micro;L||3&micro;L||8.7&micro;L||30&micro;L
Line 182: Line 214:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||9/4 spin-DT||E||S||X||P||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 189: Line 221:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||9/7 RBS-lysis1-DT||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||9/7 RBS-lysis1-DT||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts|||10&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
|2 cuts|||10&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
Line 196: Line 228:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||9/6 RBS-lysis2-DT||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||9/6 RBS-lysis2-DT||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
||2 cuts||13&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||9&micro;L||30&micro;L
||2 cuts||13&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||9&micro;L||30&micro;L
Line 203: Line 235:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||9/7 RBS-lysis3-DT||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||9/7 RBS-lysis3-DT||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts|||10&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
|2 cuts|||10&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
|-
|-
|NC||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
|NC||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
-
 
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||8/20 J23100 RBS-lacZα-DT-(1)||E||S||X||P||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
|-
|-
|NC||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
|NC||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
-
|-
 
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||8/20 RBS-lacZα-DT||E||S||X||P||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
|-
|-
|NC||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
|NC||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
-
|-
 
|}
|}
 +
</div>
===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 236: 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 attenuater+aptamer 12-1R-DT||1||10||11||Amp
+
|9/8 Pcon-pT181 attenuator+aptamer 12-1R-DT||1||10||11||Amp
|-
|-
|9/8 Plac+RBS-lysis1-DT||2||20||22||CP
|9/8 Plac+RBS-lysis1-DT||2||20||22||CP
Line 248: Line 277:
|9/8 Plac+spinach||2||20||22||CP
|9/8 Plac+spinach||2||20||22||CP
|-
|-
-
|9/8 Plac+pT181 attenuater||2||20||22||CP
+
|9/8 Plac+pT181 attenuator||2||20||22||CP
|-
|-
|9/8 Plux+RBS-lysis1-DT||1||10||11||CP
|9/8 Plux+RBS-lysis1-DT||1||10||11||CP
Line 254: 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
|-
|-
-
|9/8 Pcon-pT181 attenuater+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 271: Line 299:
|1||100bp ladder|| ||  
|1||100bp ladder|| ||  
|-
|-
-
|2||Pcon-spin-DT||E||S
+
|2||Pcon-spinach-DT||EcoRI||SpeI
|-
|-
-
|3||Pcon-spin-DT|| ||  
+
|3||Pcon-spinach-DT|| ||  
|-
|-
-
|4||Fusion1 attenuator||X||P
+
|4||Fusion1 attenuator||XbaI||PstI
|-
|-
|5||Fusion1 attenuator|| ||  
|5||Fusion1 attenuator|| ||  
|-
|-
-
|6||Fusion3m2 attenuator||X||P
+
|6||Fusion3m2 attenuator||XbaI||PstI
|-
|-
|7||Fusion3m2 attenuator|| ||  
|7||Fusion3m2 attenuator|| ||  
|-
|-
-
|8||Fusion1 anti||X||P
+
|8||Fusion1 anti||XbaI||PstI
|-
|-
|9||Fusion1 anti|| ||  
|9||Fusion1 anti|| ||  
|-
|-
-
|10||Fusion6 anti||X||P
+
|10||Fusion6 anti||XbaI||PstI
|-
|-
|11||Fusion6 anti|| ||  
|11||Fusion6 anti|| ||  
|-
|-
-
|12||aptamer 12-1M||X||P
+
|12||aptamer 12-1M||XbaI||PstI
|-
|-
|13||aptamer 12-1M|| ||  
|13||aptamer 12-1M|| ||  
|-
|-
-
|14||aptamer 12-P||X||P
+
|14||aptamer 12-P||XbaI||PstI
|-
|-
|15||aptamer 12-P|| ||
|15||aptamer 12-P|| ||
|-
|-
-
|16||spinach-DT||X||P
+
|16||spinach-DT||XbaI||PstI
|-
|-
|}
|}
 +
[[File:Igku_985.png]]
{| class="wikitable"
{| class="wikitable"
!Lane||Sample||Enzyme1||Enzyme2
!Lane||Sample||Enzyme1||Enzyme2
Line 307: Line 336:
|1||spinach-DT|| ||  
|1||spinach-DT|| ||  
|-
|-
-
|2||J23100-RBS-lacZα-DT||E||S
+
|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||E||S
+
|5||RBS-lacZ&alpha;-DT||EcoRI||SpeI
|-
|-
-
|6||RBS-lacZα-DT|| ||  
+
|6||RBS-lacZ&alpha;-DT|| ||  
|-
|-
-
|7||RBS-lysis1-DT||X||P
+
|7||RBS-lysis1-DT||XbaI||PstI
|-
|-
|8||RBS-lysis1-DT|| ||  
|8||RBS-lysis1-DT|| ||  
Line 327: Line 356:
|1||100bp ladder|| ||  
|1||100bp ladder|| ||  
|-
|-
-
|2||RBS-lysis2-DT||X||P
+
|2||RBS-lysis2-DT||XbaI||PstI
|-
|-
|3||RBS-lysis2-DT|| ||  
|3||RBS-lysis2-DT|| ||  
|-
|-
-
|4||RBS-lysis3-DT||X||P
+
|4||RBS-lysis3-DT||XbaI||PstI
|-
|-
|5||RBS-lysis3-DT|| ||  
|5||RBS-lysis3-DT|| ||  
|-
|-
-
|6||Pcon pT181 anti||S||P
+
|6||Pcon pT181 anti||SpeI||PstI
|-
|-
|7||Pcon pT181 anti|| ||  
|7||Pcon pT181 anti|| ||  
|-
|-
|}
|}
 +
[[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 350: Line 380:
|1||100bp ladder||  
|1||100bp ladder||  
|-
|-
-
|2||Pcon-spin-DT||E+S
+
|2||Pcon-spinach-DT||EcoRI+SpeI
|-
|-
-
|3||Pcon-spin-DT||E+S
+
|3||Pcon-Spinach-DT||EcoRI+SpeI
|-
|-
-
|5||Fusion 1 attenuator||X+P
+
|5||Fusion 1 attenuator||XbaI+PstI
|-
|-
-
|6||Fusion 1 attenuator||X+P
+
|6||Fusion 1 attenuator||XbaI+PstI
|-
|-
-
|8||Fusion3m2 attenuator||X+P
+
|8||Fusion3m2 attenuator||XbaI+PstI
|-
|-
-
|9||Fusion3m2 attenuator||X+P
+
|9||Fusion3m2 attenuator||XbaI+PstI
|-
|-
-
|11||Fusion 1 anti||X+P
+
|11||Fusion 1 antisense||XbaI+PstI
|-
|-
-
|12||Fusion 1 anti||X+P
+
|12||Fusion 1 antisense||XbaI+PstI
|-
|-
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:igku_987.png]]<br>
-
[[File:igku_xxafterxx.xxx]]<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
|-
|-
-
|Pcon-spin-DT(E+S)|| ||10.5||1.86||0.39
+
|Pcon-spinach-DT(EcoRI+SpeI)|| ||10.5||1.86||0.39
|-
|-
-
|Fusion 1 attenuator(X+P)|| ||7.0||1.71||0.27
+
|Fusion 1 attenuator(XbaI+PstI)|| ||7.0||1.71||0.27
|-
|-
-
|Fusion3m2 attenuator(X+P)|| ||8.6||1.75||0.47
+
|Fusion3m2 attenuator(XbaI+PstI)|| ||8.6||1.75||0.47
|-
|-
-
|Fusion 1 anti(X+P)|| ||7.6||1.87||0.43
+
|Fusion 1 antisense(XbaI+PstI)|| ||7.6||1.87||0.43
|-
|-
|}
|}
Line 386: Line 415:
|1||100bp ladder||  
|1||100bp ladder||  
|-
|-
-
|2||Fusion6 anti||X+P
+
|2||Fusion6 antisense||XbaI+PstI
|-
|-
-
|3||Fusion6 anti||X+P
+
|3||Fusion6 antisense||XbaI+PstI
|-
|-
-
|5||aptamer12_1M||X+P
+
|5||aptamer12_1M||XbaI+PstI
|-
|-
-
|6||aptamer12_1M||X+P
+
|6||aptamer12_1M||XbaI+PstI
|-
|-
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:igku_988b.png]]<br>
-
[[File:igku_xxafterxx.xxx]]<br>
+
[[File:igku_988a.png]]<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
|-
|-
-
|Fusion6 anti(X+P)|| ||5.0||1.76||0.20
+
|Fusion6 antisense(XbaI+PstI)|| ||5.0||1.76||0.20
|-
|-
-
|aptamer12_1M(X+P)|| ||3.3||2.31||0.04
+
|aptamer12_1M(XbaI+PstI)|| ||3.3||2.31||0.04
|-
|-
|}
|}
Line 410: Line 439:
|1||100bp ladder||  
|1||100bp ladder||  
|-
|-
-
|2||aptamer12_P||X+P
+
|2||aptamer12_P||XbaI+PstI
|-
|-
-
|3||aptamer12_P||X+P
+
|3||aptamer12_P||XbaI+PstI
|-
|-
-
|5||spinach-DT||E+S
+
|5||spinach-DT||EcoRI+SpeI
|-
|-
-
|6||spinach-DT||E+S
+
|6||spinach-DT||EcoRI+SpeI
|-
|-
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:igku_989b.png]]<br>
-
[[File:igku_xxafterxx.xxx]]<br>
+
[[File:igku_989a.png]]<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
|-
|-
-
|aptamer12_P(X+P)|| ||3.4||1.72||0.26
+
|aptamer12_P(XbaI+PstI)|| ||3.4||1.72||0.26
|-
|-
-
|spinach-DT(E+S)|| ||10.1||1.57||0.47
+
|spinach-DT(EcoRI+SpeI)|| ||10.1||1.57||0.47
|-
|-
|}
|}
Line 434: Line 463:
|1||100bp ladder||  
|1||100bp ladder||  
|-
|-
-
|2||J23100-RBS-laczα-DT||E+S
+
|2||J23100-RBS-lacz&alpha;-DT||EcoRI+SpeI
|-
|-
-
|3||J23100-RBS-laczα-DT||E+S
+
|3||J23100-RBS-lacz&alpha;-DT||EcoRI+SpeI
|-
|-
-
|5||RBS-laczα-DT||E+S
+
|5||RBS-lacz&alpha;-DT||EcoRI+SpeI
|-
|-
-
|6||RBS-laczα-DT||E+S
+
|6||RBS-lacz&alpha;-DT||EcoRI+SpeI
|-
|-
-
|8||RBS-lysis1-DT||X+P
+
|8||RBS-lysis1-DT||XbaI+PstI
|-
|-
-
|9||RBS-lysis1-DT||X+P
+
|9||RBS-lysis1-DT||XbaI+PstI
|-
|-
-
|11||RBS-lysis2-DT||X+P
+
|11||RBS-lysis2-DT||XbaI+PstI
|-
|-
-
|12||RBS-lysis2-DT||X+P
+
|12||RBS-lysis2-DT||XbaI+PstI
|-
|-
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:igku_9810b.png]]<br>
-
[[File:igku_xxafterxx.xxx]]<br>
+
[[File:igku_9810a.png]]<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
|-
|-
-
|Pcon-RBS-laczα-DT(E+S)|| ||5.6||2.03||0.25
+
|Pcon-RBS-lacz&alpha;-DT(EcoRI+SpeI)|| ||5.6||2.03||0.25
|-
|-
-
|RBS-laczα-DT(E+S)|| ||4.4||2.04||0.33
+
|RBS-lacz&alpha;-DT(EcoRI+SpeI)|| ||4.4||2.04||0.33
|-
|-
-
|RBS-lysis1-DT(X+P)|| ||5.9||2.22||0.53
+
|RBS-lysis1-DT(XbaI+PstI)|| ||5.9||2.22||0.53
|-
|-
-
|RBS-lysis2-DT(X+P)|| ||5.1||1.67||0.48
+
|RBS-lysis2-DT(XbaI+PstI)|| ||5.1||1.67||0.48
|-
|-
|}
|}
Line 470: Line 499:
|1||100bp ladder||  
|1||100bp ladder||  
|-
|-
-
|2||RBS-lysis3-DT||X+P
+
|2||RBS-lysis3-DT||XbaI+PstI
|-
|-
-
|3||RBS-lysis3-DT||X+P
+
|3||RBS-lysis3-DT||XbaI+PstI
|-
|-
-
|5||Pcon-pT181 anti||S+P
+
|5||Pcon-pT181 antisense||SpeI+PstI
|-
|-
-
|6||Pcon-pT181 anti||S+P
+
|6||Pcon-pT181 antisense||SpeI+PstI
|-
|-
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:igku_9811b.png]]<br>
-
[[File:igku_xxafterxx.xxx]]<br>
+
[[File:igku_9811a.png]]<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
|-
|-
-
|RBS-lysis3-DT(X+P)|| ||23.4||1.72||0.88
+
|RBS-lysis3-DT(XbaI+PstI)|| ||23.4||1.72||0.88
|-
|-
-
|Pcon-pT181 anti(S+P)|| ||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||name||xx &micro;g/mL||name||xx &micro;g/mL||xx &micro;g/mL
+
|experiment||Pcon-spinach-DT(EcoRI&SpeI)||10.5&micro;g/mL||pSB6A1(EcoRI&SpeI)||8.9&micro;g/mL
|-
|-
-
|NC||name||xx &micro;g/mL||name||xx &micro;g/mL||xx &micro;g/mL
+
|experiment||Pcon-spinach-DT(EcoRI&SpeI)||10.5&micro;g/mL||pSB4K5(EcoRI&SpeI)||21.8&micro;g/mL
-
|}
+
-
{| class="wikitable"
+
-
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
+
|-
|-
-
|experiment||name||xx &micro;g/mL||name||xx &micro;g/mL||xx &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||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||spinach-DT(XbaI+PstI)||10.1&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
|-
|-
-
|NC||name||xx &micro;g/mL||name||xx &micro;g/mL||xx &micro;g/mL
 
|}
|}
 +
</div>
 +
===PCR===
===PCR===
<!-- ここから -->
<!-- ここから -->
Line 514: Line 548:
<span class="author">No name</span>
<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
+
!KaiA(plasmid)||KOD plus||10x buffer||dNTP||MgSO4||Primer(661-RBS-KaiA-fwd)||Primer(kaiA-662-rev-ver2)||MilliQ||total
|-
|-
-
| ||0.5||2.5||2.5||1.5||0.75||0.75|| ||25
+
|1||0.5||2.5||2.5||1.5||0.75||0.75||15.5||25
|}
|}
{| class="wikitable"
{| class="wikitable"
!PreDenature||Denature||Annealing||Extension||cycle
!PreDenature||Denature||Annealing||Extension||cycle
|-
|-
-
|　&deg;C||　&deg;C||　&deg;C||　&deg;C||--
+
|94&deg;C||98&deg;C||57&deg;C||68&deg;C||--
|-
|-
-
|（◎Д◎）　||　||　||　||
+
|2min||10s||30s||28s||30cycles
|}
|}
-
===Gel Extraction===
 
-
<!-- こっから -->
 
-
<div class="experiment">
 
-
<span class="author">じっけんしゃ</span>
 
{| class="wikitable"
{| class="wikitable"
-
!Lane||DNA||Enzyme
+
!KaiB(plasmid)||KOD plus||10x buffer||dNTP||MgSO4||Primer(662-RBS-KaiB-fwd-ver2)||Primer(kaiB-663-rev-ver2)||MilliQ||total
|-
|-
-
|（＞ω・）b || ||
+
|1||0.5||2.5||2.5||1.5||0.75||0.75||15.5||25
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
|-
|-
-
| || ||
+
|94&deg;C||98&deg;C||57&deg;C||68&deg;C||--
 +
|-
 +
|2min||10s||30s||28s||30cycles
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
 
-
[[File:igku_xxafterxx.xxx]]<br>
 
{| class="wikitable"
{| class="wikitable"
-
!Name||quantity||concentration[&micro;g/mL]||260/280||260/230
+
!DT(plasmid)||KOD plus||10x buffer||dNTP||MgSO4||Primer(664-DT-fwd-ver2)||Primer(DT-suffix-660-rev-ver2)||MilliQ||total
|-
|-
-
|（´ω｀）|| || || ||
+
|0.1||0.5||2.5||2.5||1.5||0.75||0.75||16.4||25
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
|-
|-
-
|（ΦωΦ^）|| || || ||
+
|94&deg;C||98&deg;C||57&deg;C||68&deg;C||--
 +
|-
 +
|2min||10s||30s||28s||30cycles
|}
|}
{| class="wikitable"
{| class="wikitable"
-
!Lane||DNA||Enzyme
+
!P-KaiB(PCR prpduct)||KOD plus||10x buffer||dNTP||MgSO4||Primer(prefix-PkaiBC-fwd-ver2)||Primer(PkaiBC-suffix--662-rev-ver2)||MilliQ||total
|-
|-
-
|（＞ω・）b || ||
+
|0.7||0.5||2.5||2.5||1.5||0.75||0.75||15.8||25
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
|-
|-
-
| || ||
+
|94&deg;C||98&deg;C||57&deg;C||68&deg;C||--
 +
|-
 +
|2min||10s||30s||28s||30cycles
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
 
-
[[File:igku_xxafterxx.xxx]]<br>
 
{| class="wikitable"
{| class="wikitable"
-
!Name||quantity||concentration[&micro;g/mL]||260/280||260/230
+
!Plac(1)(plasmid)||KOD plus||10x buffer||dNTP||MgSO4||Primer(GG0-pSBIC3-fwd-ver2)||Primer(Plac-GG1-rev-ver2)||MilliQ||total
 +
|-
 +
|0.13||0.5||2.5||2.5||1.5||0.75||0.75||16.37||25
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||98&deg;C||57&deg;C||68&deg;C||--
 +
|-
 +
|2min||10s||30s||60s||30cycles
 +
|}
 +
{| class="wikitable"
 +
!KaiC(plasmid)||KOD plus||10x buffer||dNTP||MgSO4||Primer(GG3-RBS-KaiC-fwd-ver2)||Primer(KaiC-GG4-rev-ver2)||MilliQ||total
 +
|-
 +
|1||0.5||2.5||2.5||1.5||0.75||0.75||15.5||25
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||98&deg;C||57&deg;C||68&deg;C||--
 +
|-
 +
|2min||10s||30s||60s||30cycles
 +
|}
 +
 
 +
===Gel Extraction===
 +
<div class="experiment">
 +
<span class="author">Yoshida</span>
 +
{| class="wikitable"
 +
!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
 +
|-
 +
|}
 +
[[File:9898.jpg]]<br>
 +
[[File:9897.jpg]]<br>
 +
{| class="wikitable"
 +
!Lane||DNA
 +
|-
 +
|1||Plac
 +
|-
 +
|2||Plac
 +
|-
 +
|3||KaiC
 +
|-
 +
|4||KaiC
 +
|-
 +
|5||nega
|-
|-
-
|（´ω｀）|| || || ||
+
|6||1kb ladder
|-
|-
-
|（ΦωΦ^）|| || || ||
 
|}
|}
 +
[[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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