Template:Kyoto/Notebook/Sep 10

From 2013.igem.org

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Line 1: Line 1:
==Sep 10==
==Sep 10==
===Ligation===
===Ligation===
-
 
-
<!-- こっから -->
 
<div class="experiment">
<div class="experiment">
<span class="author">Nakamoto</span>
<span class="author">Nakamoto</span>
Line 8: Line 6:
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
|-
|-
-
|experiment||9/6 Pcon-RBS-luxR-DT||1.9&micro;L||9/6Plux-RBS-GFP-DT||22 &micro;L|| &micro;L
+
|experiment||9/6 Pcon-RBS-luxR-DT||1.9&micro;L||9/6Plux-RBS-GFP-DT||22 &micro;L||3.5&micro;L
|-
|-
-
|experiment||9/6 Plux||2&micro;L||9/8 RBS-lysis1-DT||6.6&micro;L||xx &micro;L
+
|experiment||9/6 Plux||2&micro;L||9/8 RBS-lysis1-DT||6.6&micro;L||3.5&micro;L
|-
|-
-
|experiment||9/6 Plux||2&micro;L||9/8 RBS-lysis2-DT||6.3&micro;L||xx &micro;L
+
|experiment||9/6 Plux||2&micro;L||9/8 RBS-lysis2-DT||6.3&micro;L||3.5&micro;L
|-
|-
-
|experiment||9/8 Ptet-PT181 antisense||1.6&micro;L||9/8 spinach-DT||4.4&micro;L||xx &micro;L
+
|experiment||9/8 Ptet-PT181 antisense||1.6&micro;L||9/8 spinach-DT||4.4&micro;L||3.0&micro;L
|-
|-
-
|experiment||9/8 Pcon-PT181 attenuator||3.4&micro;L||9/8 apz 12-1R-PT||2.1&micro;L||xx &micro;L
+
|experiment||9/8 Pcon-PT181 attenuator||3.4&micro;L||9/8 apz 12-1R-PT||2.1&micro;L||2.8&micro;L
|-
|-
-
|experiment||9/8 Pcon||3.4&micro;L||9/8 apz 12-1R-PT||2.3&micro;L||xx &micro;L
+
|experiment||9/8 Pcon||3.4&micro;L||9/8 apz 12-1R-PT||2.3&micro;L||2,9&micro;L
|-
|-
-
|experiment||9/8 DT||3.0&micro;L||9/8 Pcon-PT181 attenuator||7.9&micro;L||xx &micro;L
+
|experiment||9/8 DT||3.0&micro;L||9/8 Pcon-PT181 attenuator||7.9&micro;L||3.5&micro;L
|-
|-
-
|experiment||9/10 pSB1C3||1.8&micro;L||9/8 F3m2 attenuator||4.6&micro;L||xx &micro;L
+
|experiment||9/10 pSB1C3||1.8&micro;L||9/8 F3m2 attenuator||4.6&micro;L||3.2&micro;L
|-
|-
-
|experiment||9/10 pSB1C3||1.8&micro;L||9/8 F1 attenuator||3.4&micro;L||xx &micro;L
+
|experiment||9/10 pSB1C3||1.8&micro;L||9/8 F1 attenuator||3.4&micro;L||2.6&micro;L
|}
|}
</div>
</div>
-
<!-- ここまでをコピーしてね -->
 
===Miniprep===
===Miniprep===
Line 51: Line 48:
<span class="author">Nakamoto</span>
<span class="author">Nakamoto</span>
{| class="wikitable"
{| class="wikitable"
-
! ||9/10　Plux-RBS-GFP-DT||EcoR1||Spel||Buffer||BSA||MilliQ||total
+
! ||9/10　Plux-RBS-GFP-DT||EcoRI||SpeI||Buffer||BSA||MilliQ||total
|-
|-
|2cuts||12.1&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||9.9&micro;L||30&micro;L
|2cuts||12.1&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||9.9&micro;L||30&micro;L
Line 58: Line 55:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||9/10　Spinach-DT||Xbal||Pstl||Buffer||BSA||MilliQ||total
+
! ||9/10　Spinach-DT||XbaI||PstI||Buffer||BSA||MilliQ||total
|-
|-
|2cuts||9.9&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||12.1&micro;L||30&micro;L
|2cuts||9.9&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||12.1&micro;L||30&micro;L
Line 65: Line 62:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||9/6 apt12-1R -DT||Xbal||Pstl||Buffer||BSA||MilliQ||total
+
! ||9/6 apt12-1R -DT||XbaI||PstI||Buffer||BSA||MilliQ||total
|-
|-
|2cuts||8.0&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||14.0&micro;L||30&micro;L
|2cuts||8.0&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||14.0&micro;L||30&micro;L
Line 72: Line 69:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||9/10　Pcon-pt81 attenuator ||EcoR1||Spel||Buffer||BSA||MilliQ||total
+
! ||9/10　Pcon-pt81 attenuator ||EcoRI||SpeI||Buffer||BSA||MilliQ||total
|-
|-
|2cuts||9.8&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||12.2&micro;L||30&micro;L
|2cuts||9.8&micro;L||1&micro;L||1&micro;L||3&micro;L||3&micro;L||12.2&micro;L||30&micro;L
Line 89: Line 86:
|1||100bp ladder||--||--
|1||100bp ladder||--||--
|-
|-
-
|2|| Plux-RBS-GEP-DT||EcoR1||Spel
+
|2|| Plux-RBS-GEP-DT||EcoRI||SpeI
|-
|-
|3|| Plux-RBS-GEP-DT||--||--
|3|| Plux-RBS-GEP-DT||--||--
|-
|-
-
|4||Spinach-DT||Xbal||Pstl
+
|4||Spinach-DT||XbaI||PstI
|-
|-
|5||Spinach-DT||--||--
|5||Spinach-DT||--||--
|-
|-
-
|6||aptamer 121R-DT||Xbal||Pstl
+
|6||aptamer 121R-DT||XbaI||PstI
|-
|-
|7||aptamer 121R-DT||--||--
|7||aptamer 121R-DT||--||--
|-
|-
-
|8||Pcon-pt181 attenuator|||EcoR1||Spel
+
|8||Pcon-pt181 attenuator|||EcoRI||SpeI
|-
|-
|9|| Pcon-pt181 attenuator||--||--
|9|| Pcon-pt181 attenuator||--||--
Line 178: Line 175:
|}
|}
</div>
</div>
 +
[[File:Igku_0910_E3.jpg]]
===Gel Extraction===
===Gel Extraction===
Line 232: Line 230:
|}
|}
</div>
</div>
-
<!-- ここまでをコピーしてね -->
+
[[File:Igku_0910_G3.jpg]]
===Liquid Culture===
===Liquid Culture===
Line 369: Line 367:
<span class="author">Nakamoto</span>
<span class="author">Nakamoto</span>
{| class="wikitable"
{| class="wikitable"
-
!Name||Sample||Competent Cells||Total||Plate
+
!Name||Plate
|-
|-
-
|F1 attenuator +p3B1C3||&micro;L||&micro;L||&micro;L||CP
+
|F1 attenuator +p3B1C3||CP
|-
|-
-
|F3n2 attenuator +p3B1C3||&micro;L||&micro;L||&micro;L||CP
+
|F3n2 attenuator +p3B1C3||CP
|-
|-
-
|F1 antisense +p3B1C3||&micro;L||&micro;L||&micro;L||CP
+
|F1 antisense +p3B1C3||CP
|-
|-
-
|F6 antisense+p3B1C3||&micro;L||&micro;L||&micro;L||CP
+
|F6 antisense+p3B1C3||CP
|-
|-
-
|apt12-P +p3B1C3||&micro;L||&micro;L||&micro;L||CP
+
|apt12-P +p3B1C3||CP
|-
|-
-
|apt12-1M +p3B1C3||&micro;L||&micro;L||&micro;L||CP
+
|apt12-1M +p3B1C3||CP
|-
|-
-
|Pcon-luxR+Plux-GFP||&micro;L||&micro;L||&micro;L||Amp
+
|Pcon-luxR+Plux-GFP||Amp
|-
|-
-
|Plux+RBS-lysis1-DT||&micro;L||&micro;L||&micro;L||CP
+
|Plux+RBS-lysis1-DT||CP
|-
|-
-
|Plux+RBS-lysis2-DT||&micro;L||&micro;L||&micro;L||CP
+
|Plux+RBS-lysis2-DT||CP
|-
|-
-
|Ptet-pT181antisense+Spinach-DT||&micro;L||&micro;L||&micro;L||CP
+
|Ptet-pT181antisense+Spinach-DT||CP
|-
|-
-
|Pcon-pT181 attenuator+apt121R-DT||&micro;L||&micro;L||&micro;L||Amp
+
|Pcon-pT181 attenuator+apt121R-DT||Amp
|-
|-
-
|Pcon-1 apt12-1R-DT||&micro;L||&micro;L||&micro;L||Amp
+
|Pcon-1 apt12-1R-DT||Amp
|-
|-
-
|DT+Pcon-pT181 attenuator||&micro;L||&micro;L||&micro;L||CP
+
|DT+Pcon-pT181 attenuator||CP
|}
|}
</div>
</div>
-
<!-- ここまでをコピーしてね -->
 
===Electrophoresis===
===Electrophoresis===
Line 410: Line 407:
|1||100bp ladder||--||--
|1||100bp ladder||--||--
|-
|-
-
|2||Spinach-DT||Xbal||Ptsl
+
|2||Spinach-DT||XbaI||PstI
|-  
|-  
-
|3||Spinach-DT||Xbal||Ptsl
+
|3||Spinach-DT||XbaI||PstI
|-  
|-  
-
|4||Spinach-DT||Xbal||Ptsl
+
|4||Spinach-DT||XbaI||PstI
|-  
|-  
-
|5||Spinach-DT||Xbal||Ptsl
+
|5||Spinach-DT||XbaI||PstI
|-
|-
|6||100bp ladder||--||--  
|6||100bp ladder||--||--  
Line 427: Line 424:
|1||100bp ladder||--||--
|1||100bp ladder||--||--
|-
|-
-
|2||Pcon-pT181 attenuator||EcoR1||Spel
+
|2||Pcon-pT181 attenuator||EcoRI||SpeI
|-  
|-  
-
|3||Pcon-pT181 attenuator||EcoR1||Spel
+
|3||Pcon-pT181 attenuator||EcoRI||SpeI
|-  
|-  
-
|4||Pcon-pT181 attenuator||EcoR1||Spel
+
|4||Pcon-pT181 attenuator||EcoRI||Spei
  |-  
  |-  
-
|5||Pcon-pT181 attenuator||EcoR1||Spel
+
|5||Pcon-pT181 attenuator||EcoRI||SpeI
|-
|-
|6||100bp ladder||--||--  
|6||100bp ladder||--||--  
Line 442: Line 439:
!Lane||Sample||Enzyme1||Enzyme2
!Lane||Sample||Enzyme1||Enzyme2
|-
|-
-
|1||Plux-RBS-GFP-DT||EcoR1||Spel
+
|1||Plux-RBS-GFP-DT||EcoRI||SpeI
|-
|-
-
|2||Plux-RBS-GFP-DT ||EcoR1||Spel
+
|2||Plux-RBS-GFP-DT ||EcoRI||SpeI
|-  
|-  
-
|3||Plux-RBS-GFP-DT ||EcoR1||Spel
+
|3||Plux-RBS-GFP-DT ||EcoRI||SpeI
|-  
|-  
-
|4||Plux-RBS-GFP-DT ||EcoR1||Spel
+
|4||Plux-RBS-GFP-DT ||EcoRI||SpeI
  |-  
  |-  
|5||100bp ladder||--||--
|5||100bp ladder||--||--
|-
|-
-
|6||apt12-1R-DT||Xbal||Pstl
+
|6||apt12-1R-DT||XbaI||PstI
|-  
|-  
-
|7||apt12-1R-DT||Xbal||Pstl
+
|7||apt12-1R-DT||XbaI||PstI
|-  
|-  
-
|8||apt12-1R-DT||Xbal||Pstl
+
|8||apt12-1R-DT||XbaI||PstI
|-
|-
-
|9||apt12-1R-DT||Xbal||Pstl
+
|9||apt12-1R-DT||XbaI||PstI
|}
|}
[[File:igku_91012.jpg]]<br>
[[File:igku_91012.jpg]]<br>

Latest revision as of 18:06, 27 September 2013

Contents

Sep 10

Ligation

Nakamoto

stateVectorInserterLigation High ver.2
experiment9/6 Pcon-RBS-luxR-DT1.9µL9/6Plux-RBS-GFP-DT22 µL3.5µL
experiment9/6 Plux2µL9/8 RBS-lysis1-DT6.6µL3.5µL
experiment9/6 Plux2µL9/8 RBS-lysis2-DT6.3µL3.5µL
experiment9/8 Ptet-PT181 antisense1.6µL9/8 spinach-DT4.4µL3.0µL
experiment9/8 Pcon-PT181 attenuator3.4µL9/8 apz 12-1R-PT2.1µL2.8µL
experiment9/8 Pcon3.4µL9/8 apz 12-1R-PT2.3µL2,9µL
experiment9/8 DT3.0µL9/8 Pcon-PT181 attenuator7.9µL3.5µL
experiment9/10 pSB1C31.8µL9/8 F3m2 attenuator4.6µL3.2µL
experiment9/10 pSB1C31.8µL9/8 F1 attenuator3.4µL2.6µL

Miniprep

Nakamoto

DNAconcentration[µg/mL]260/280260/230
spinach-DT202.41.971.93
Plux-RBS-GFP-DT164.22.802.06
Pcon-DT attenuator205.41.812.39
RSBJC3-2240.81.731.43
Plaz-1240.81.731.43

Restriction Enzyme Digestion

Nakamoto

9/10　Plux-RBS-GFP-DTEcoRISpeIBufferBSAMilliQtotal
2cuts12.1µL1µL1µL3µL3µL9.9µL30µL
NC0.6µL0µL0µL1µL1µL7.4µL10µL
9/10　Spinach-DTXbaIPstIBufferBSAMilliQtotal
2cuts9.9µL1µL1µL3µL3µL12.1µL30µL
NC0.5µL0µL0µL1µL1µL7.5µL10µL
9/6 apt12-1R -DTXbaIPstIBufferBSAMilliQtotal
2cuts8.0µL1µL1µL3µL3µL14.0µL30µL
NC0.4µL0µL0µL1µL1µL7.6µL10µL
9/10　Pcon-pt81 attenuator EcoRISpeIBufferBSAMilliQtotal
2cuts9.8µL1µL1µL3µL3µL12.2µL30µL
NC0.5µL0µL0µL1µL1µL7.5µL10µL

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2 Plux-RBS-GEP-DTEcoRISpeI
3 Plux-RBS-GEP-DT----
4Spinach-DTXbaIPstI
5Spinach-DT----
6aptamer 121R-DTXbaIPstI
7aptamer 121R-DT----
8Pcon-pt181 attenuatorEcoRISpeI
9 Pcon-pt181 attenuator----
10100bp ladder----

Igku 9104.jpg

Colony PCR

Tatsui

Samplebase pair
9/9 Pcon-pT181 antisense--
9/8 J223100(entA)--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min10sec30

Electrophoresis

Tatsui

LaneSample
1100bp ladder
2Pcon Pt181 antisense1+spinach-DT
3Pcon Pt181 antisense2+spinach-DT
4Pcon Pt181 antisense3+spinach-DT
5Pcon Pt181 antisense4+spinach-DT
6Pcon Pt181 antisense5+spinach-DT
7Pcon Pt181 antisense6+spinach-DT
8Pcon Pt181 antisense7+spinach-DT
9Pcon Pt181 antisense8+spinach-DT
10Pcon Pt181 antisense9+spinach-DT
11Pcon Pt181 antisense10+spinach-DT
12Pcon Pt181 antisense11+spinach-DT
13Pcon Pt181 antisense12+spinach-DT
14100bp ladder

Igku 0910 E2.jpg

LaneSample
11kbp ladder
2Pcon entA1
3Pcon entA2
4Pcon entA3
51kbp ladder

Igku 0910 E3.jpg

Gel Extraction

Tatsui

LaneDNAEnzyme
11kbp ladder--
2KaiA(200pg)--
3KaiA(200pg)--
4----
5KaiA(400pg)--
6KaiA(400pg)--
7----
8KaiA(800pg--
9KaiA(800pg)--
101kbp ladder--

Igku 9101.jpg
Igku 9102.jpg

LaneDNAEnzyme
11kbp ladder--
2PKaiBC(200pg)--
3PKaiBC(200pg)--
4----
5PKaiBC(400pg)--
6PKaiBC(400pg)--
7----
8PKaiBC(800pg--
9PKaiBC(800pg)--
101kbp ladder--

Igku 0910 G3.jpg

Liquid Culture

Hirano

Samplemedium
9/4 J23100①(JM109)LB(Amp)
9/8 J23100① (entA)LB(Amp)　

Gel Extraction

Tatsui

LaneDNAEnzyme
11kbp ladder--
2KaiC--
3KaiC--
4KaiC--
5----
61kbp ladder--
71kbp ladder--
8KaiC--
9KaiC--
10KaiC--
11----
121kbp ladder--

Igku 9105.jpg
Igku 9106.jpg

Liquid Culture

Tatsui

Samplemedium
9/10 Pcon-pt181 antisense-Spinach-DTPlusgrow 　

Colony PCR

No name

Samplebase pair
9/9 Phad larac-1 RBS-lux1-DT2331
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s2min30
Samplebase pair
9/9 Pcon-lacZα (psB4KS)712
9/9 Pcon-Spinach-DT(psB4KS)605
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s42s30
Samplebase pair
9/9 Plac+RBS-lysis3}-DT1273
9/9 Pcon+RBS-lux1-DT1079
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min12s30

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder----
29/9 Pcou-RBS-lacZα-DT(p3B4KS9)-1----
39/9 Pcou-RBS-lacZα-DT(p3B4KS9)-2----
49/9 Pcou-Spinach-DT(pB4KS)-1----
59/9 Pcou-Spinach-DT(pB4KS)-2----
6100bp ladder----

Igku 91014.jpg

Transformation

Nakamoto

NamePlate
F1 attenuator +p3B1C3CP
F3n2 attenuator +p3B1C3CP
F1 antisense +p3B1C3CP
F6 antisense+p3B1C3CP
apt12-P +p3B1C3CP
apt12-1M +p3B1C3CP
Pcon-luxR+Plux-GFPAmp
Plux+RBS-lysis1-DTCP
Plux+RBS-lysis2-DTCP
Ptet-pT181antisense+Spinach-DTCP
Pcon-pT181 attenuator+apt121R-DTAmp
Pcon-1 apt12-1R-DTAmp
DT+Pcon-pT181 attenuatorCP

Electrophoresis

Okazaki

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2Spinach-DTXbaIPstI
3Spinach-DTXbaIPstI
4Spinach-DTXbaIPstI
5Spinach-DTXbaIPstI
6100bp ladder----

Igku 9108.jpg
Igku 9109.jpg

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2Pcon-pT181 attenuatorEcoRISpeI
3Pcon-pT181 attenuatorEcoRISpeI
4Pcon-pT181 attenuatorEcoRISpei
5Pcon-pT181 attenuatorEcoRISpeI
6100bp ladder----

Igku 91010.jpg
Igku 91011.jpg

LaneSampleEnzyme1Enzyme2
1Plux-RBS-GFP-DTEcoRISpeI
2Plux-RBS-GFP-DT EcoRISpeI
3Plux-RBS-GFP-DT EcoRISpeI
4Plux-RBS-GFP-DT EcoRISpeI
5100bp ladder----
6apt12-1R-DTXbaIPstI
7apt12-1R-DTXbaIPstI
8apt12-1R-DTXbaIPstI
9apt12-1R-DTXbaIPstI

Igku 91012.jpg
Igku 91013.jpg

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