Template:Kyoto/Notebook/Aug 27

From 2013.igem.org

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==Aug 27==
==Aug 27==
===Restriction Enzyme Digestion===
===Restriction Enzyme Digestion===
-
<!-- こっから -->
 
<div class="experiment">
<div class="experiment">
<span class="author">No name</span>
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
-
!8/21 pT181 attenuator-1(330&micro;g/mL)||EcoRI||SpeI||10x BufferB||100x BSA||MilliQ||total
+
! ||8/21 pT181 attenuator-1(330&micro;g/mL)||EcoRI||SpeI||10x BufferB||100x BSA||MilliQ||total
|-
|-
-
|3.0&micro;L||0.5&micro;L||0.5&micro;L||3.0&micro;L||0.3&micro;L||22.7&micro;L||30&micro;L
+
|2 cuts||3.0&micro;L||0.5&micro;L||0.5&micro;L||3.0&micro;L||0.3&micro;L||22.7&micro;L||30&micro;L
|-
|-
-
|0.6&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.3&micro;L||10&micro;L
+
|NC||0.6&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.3&micro;L||10&micro;L
|}
|}
{| class="wikitable"
{| class="wikitable"
-
!8/17 DT-1(188&micro;g/mL)||EcoRI||XbaI||10x BufferB||100x BSA||MilliQ||total
+
! ||8/17 DT-1(188&micro;g/mL)||EcoRI||XbaI||10x BufferB||100x BSA||MilliQ||total
|-
|-
-
|3.0&micro;L||0.5&micro;L||0.5&micro;L||3.0&micro;L||0.3&micro;L||22.7&micro;L||30&micro;L
+
|2 cuts||3.0&micro;L||0.5&micro;L||0.5&micro;L||3.0&micro;L||0.3&micro;L||22.7&micro;L||30&micro;L
|-
|-
-
|1.0&micro;l||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||7.9&micro;L||10&micro;L
+
|NC||1.0&micro;l||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||7.9&micro;L||10&micro;L
|}
|}
{| class="wikitable"
{| class="wikitable"
-
!8/20 Pcon-RBS-luxR-DT-2(344&micro;g/mL)||EcoRI||SpeI||10x BufferB||100x BSA||MilliQ||total
+
! ||8/20 Pcon-RBS-luxR-DT-2(344&micro;g/mL)||EcoRI||SpeI||10x BufferB||100x BSA||MilliQ||total
|-
|-
-
|2.9&micro;L||0.5&micro;L||0.5&micro;L||3.0&micro;L||0.3&micro;L||22.8&micro;L||30&micro;L
+
|2 cuts||2.9&micro;L||0.5&micro;L||0.5&micro;L||3.0&micro;L||0.3&micro;L||22.8&micro;L||30&micro;L
|-
|-
-
|0.6&micro;l||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.3&micro;L||10&micro;L
+
|NC||0.6&micro;l||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.3&micro;L||10&micro;L
|}
|}
</div>
</div>
 +
===Electrophoresis===
===Electrophoresis===
<div class="experiment">
<div class="experiment">
Line 48: Line 48:
|8||100bp ladder||--||--
|8||100bp ladder||--||--
|}
|}
 +
[[File:Igku Aug27 Electrophoresis(N1)-1.jpg]]<br>
 +
===Colony PCR===
===Colony PCR===
<!-- ここから -->
<!-- ここから -->
Line 68: Line 70:
|5min||30s||30s||3min||30cycles
|5min||30s||30s||3min||30cycles
|}
|}
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1||100bp ladder
 +
|-
 +
|2||P&lambda;-luxI-(1)
 +
|-
 +
|3||P&lambda;-luxI-(2)
 +
|-
 +
|4||NC
 +
|}
 +
[[File:Igku Aug27 Electrophoresis(ColoP)(N3)-2.jpg]]<br>
 +
</div>
 +
</div>
</div>
 +
===Liquid Culture===
===Liquid Culture===
<!-- ここから -->
<!-- ここから -->
Line 80: Line 97:
|8/26 Pλ-RBS-luxI-DT-2||Plusgrow medium (+Amp)　
|8/26 Pλ-RBS-luxI-DT-2||Plusgrow medium (+Amp)　
|}
|}
 +
</div>
 +
 +
===Restriction Enzyme Digestion===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
! ||8/21 pT181 attenuator-1(330&micro;g/mL)||EcoRI||SpeI||10x BufferB||100x BSA||MilliQ||total
 +
|-
 +
|2 cuts||3.0&micro;L||0.5&micro;L||0.5&micro;L||3.0&micro;L||0.3&micro;L||22.7&micro;L||30&micro;L
 +
|-
 +
|NC||0.6&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.3&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
! ||8/17 DT-1(188&micro;g/mL)||EcoRI||XbaI||10x BufferB||100x BSA||MilliQ||total
 +
|-
 +
|2 cuts||3.0&micro;L||0.5&micro;L||0.5&micro;L||3.0&micro;L||0.3&micro;L||22.7&micro;L||30&micro;L
 +
|-
 +
|NC||1.0&micro;l||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||7.9&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
! ||8/20 Pcon-RBS-luxR-DT-2(344&micro;g/mL)||EcoRI||SpeI||10x BufferB||100x BSA||MilliQ||total
 +
|-
 +
|2 cuts||2.9&micro;L||0.5&micro;L||0.5&micro;L||3.0&micro;L||0.3&micro;L||22.8&micro;L||30&micro;L
 +
|-
 +
|NC||0.6&micro;l||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.3&micro;L||10&micro;L
 +
|}
 +
</div>
 +
===Electrophoresis===
===Electrophoresis===
<!-- こっから -->
<!-- こっから -->
Line 87: Line 132:
!Lane||Sample||Enzyme1||Enzyme2
!Lane||Sample||Enzyme1||Enzyme2
|-
|-
-
|1||100bp ladder||--||--
+
|||100bp ladder||--||--
|-
|-
-
|2||pT181 attenuator||EcoR1||SpeI
+
|1||pT181 attenuator||EcoRI||SpeI
|-
|-
-
|3||pT181 attenuator||--||--
+
|2||pT181 attenuator||--||--
|-
|-
-
|4||DT-(1)||EcoRI||XbaI
+
|3||DT-(1)||EcoRI||XbaI
|-
|-
-
|5||DT-(1)||--||--
+
|4||DT-(1)||--||--
|-
|-
-
|6||Pcon-RBS-luxR-DT-(2)||EcoRI||SpeI
+
|5||Pcon-RBS-luxR-DT-(2)||EcoRI||SpeI
|-
|-
-
|7||Pcon-RBS-luxR-DT-(2)||--||--
+
|6||Pcon-RBS-luxR-DT-(2)||--||--
|-
|-
-
|8||100bp ladder||--||--
+
|||100bp ladder||--||--
|}
|}
-
[[File:igku_xxxxxx.xxx]]<br>
+
[[File:Igku Aug27 Electrophoresis(N2)-2.jpg]]<br>
</div>
</div>
 +
===Gel Extraction===
===Gel Extraction===
<!-- こっから -->
<!-- こっから -->
Line 128: Line 174:
|8||100bpladder||--
|8||100bpladder||--
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:Igku Aug27 Gel Extraction(N3)-1-3.jpg]]<br>
-
[[File:igku_xxafterxx.xxx]]<br>
+
[[File:Igku Aug27 Gel Extraction(N4)pic(1).jpg]]<br>
 +
 
{| class="wikitable"
{| class="wikitable"
-
!Name||quantity||concentration[&micro;g/mL]||260/280||260/230
+
!Name||concentration[&micro;g/mL]||260/280||260/230
|-
|-
-
|pT181 attenuator-1(EcoRI&SpeI)||4.2||1.62||0.29||
+
|pT181 attenuator-1(EcoRI&SpeI)||4.2||1.62||0.29
 +
|-
 +
|DT-1(EcoRI&XbaI)||17||1.88||0.88
 +
|}
 +
</div>
 +
 
 +
===Transformation===
 +
<div class="experiment">
 +
<span class="author">Kojima and Hirano</span>
 +
{| class="wikitable"
 +
!Name||Sample||Competent Cells||Total||Plate
 +
|-
 +
|8/26 tetR aptamer 12_1R-DT||1&micro;L||10&micro;L||11&micro;L||CP
 +
|-
 +
|8/26 pT181 attenuator-DT||1&micro;L||10&micro;L||11&micro;L||CP
 +
|-
 +
|8/26 pT181 antisense-DT||1&micro;L||10&micro;L||11&micro;L||CP
 +
|-
 +
|8/26 Spinach-DT||1&micro;L||10&micro;L||11&micro;L||CP
 +
|-
 +
|8/26 Plac-pT181 attenuator||1&micro;L||10&micro;L||11&micro;L||CP
 +
|-
 +
|8/26 Pcon-pT181 attenuator||1&micro;L||10&micro;L||11&micro;L||Amp
 +
|-
 +
|8/26 Plac-pT181 antisense||1&micro;L||10&micro;L||11&micro;L||CP
 +
|-
 +
|8/26 Pcon-pT181 antisense||1&micro;L||10&micro;L||11&micro;L||Amp
 +
|}
 +
</div>
 +
 
 +
===Liquid culture===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Sample||medium
 +
|-
 +
|8/16 Plux||Plusgrow medium(+CP)
 +
|}
 +
 
 +
===Genome PCR===
 +
<div class="experiment">
 +
<span class="author">Yoshdia and Nakagawa</span>
 +
{| class="wikitable"
 +
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||SasA_fwd primer||SasA_rev primer||MilliQ||total
 +
|-
 +
|0.5||0.5||2.5||2.5||1.5||0.75||0.75||16||25
 +
|}
 +
{| class="wikitable"
 +
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||RpaA_fwd primer||RpaA_rev primer||MilliQ||total
 +
|-
 +
|0.5||0.5||2.5||2.5||1.5||0.75||0.75||16||25
 +
|}
 +
{| class="wikitable"
 +
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||RpaB_fwd primer||RpaB_rev primer||MilliQ||total
 +
|-
 +
|0.5||0.5||2.5||2.5||1.5||0.75||0.75||16||25
 +
|}
 +
{| class="wikitable"
 +
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||PkaiBC_fwd primer||PkaiBC_rev primer||MilliQ||total
 +
|-
 +
|0.5||0.5||2.5||2.5||1.5||0.75||0.75||16||25
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||98&deg;C||50&deg;C||68&deg;C||--
 +
|-
 +
|2 min||10 sec||30 sec||38 sec||30 cycles
 +
|}
 +
 
 +
</div>
 +
 
 +
===Ligation===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!state||colspan="2"|Vector||colspan="2"|Inserter
 +
|-
 +
|experiment||8/27 DT (EcoRI & XbaI)||2.8||8/21 RBS-lysis3 (EcoRI & SpeI)
 +
|-
 +
|experiment||8/27 DT (EcoRI & XbaI)||2.8||8/27 pT181 attenuator (EcoRI & SpeI)
 +
|}
 +
*Samples were evaporeted used evaporator into about 3 &micro;L.
 +
{| class="wikitable"
 +
!sample||MilliQ||Ligation High||total
|-
|-
-
|DT-1(EcoRI&XbaI)||17||1.88||0.88||
+
|3||4||3.5||10.5
|}
|}
 +
*incubate overnight at 4 &deg;C
</div>
</div>
-
<!-- ここまでをコピーしてね -->
 

Latest revision as of 00:47, 27 September 2013

Contents

Aug 27

Restriction Enzyme Digestion

No name

8/21 pT181 attenuator-1(330µg/mL)EcoRISpeI10x BufferB100x BSAMilliQtotal
2 cuts3.0µL0.5µL0.5µL3.0µL0.3µL22.7µL30µL
NC0.6µL0µL0µL1µL0.1µL8.3µL10µL
8/17 DT-1(188µg/mL)EcoRIXbaI10x BufferB100x BSAMilliQtotal
2 cuts3.0µL0.5µL0.5µL3.0µL0.3µL22.7µL30µL
NC1.0µl0µL0µL1µL0.1µL7.9µL10µL
8/20 Pcon-RBS-luxR-DT-2(344µg/mL)EcoRISpeI10x BufferB100x BSAMilliQtotal
2 cuts2.9µL0.5µL0.5µL3.0µL0.3µL22.8µL30µL
NC0.6µl0µL0µL1µL0.1µL8.3µL10µL

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder----
28/21 pT181 attenuatorEcoISpeI
38/21 pT181 atteniator----
48/17 DT-1EcoRIXbaI
58/17 DT-1----
68/20 Pcon-RBS-luxR-DT-2EcoISpeI
78/20 Pcon-RBS-luxR-DT-2----
8100bp ladder----

Igku Aug27 Electrophoresis(N1)-1.jpg

Colony PCR

Kojima and Yoshida

Samplebase pair
8/26 Pλ-luxI(1)
8/26 Pλ-luxI(2)
NC
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s3min30cycles
LaneSample
1100bp ladder
2Pλ-luxI-(1)
3Pλ-luxI-(2)
4NC

Igku Aug27 Electrophoresis(ColoP)(N3)-2.jpg

Liquid Culture

No name

Samplemedium
8/26 Pλ-RBS-luxI-DT-1Plusgrow medium (+Amp)
8/26 Pλ-RBS-luxI-DT-2Plusgrow medium (+Amp)　

Restriction Enzyme Digestion

No name

8/21 pT181 attenuator-1(330µg/mL)EcoRISpeI10x BufferB100x BSAMilliQtotal
2 cuts3.0µL0.5µL0.5µL3.0µL0.3µL22.7µL30µL
NC0.6µL0µL0µL1µL0.1µL8.3µL10µL
8/17 DT-1(188µg/mL)EcoRIXbaI10x BufferB100x BSAMilliQtotal
2 cuts3.0µL0.5µL0.5µL3.0µL0.3µL22.7µL30µL
NC1.0µl0µL0µL1µL0.1µL7.9µL10µL
8/20 Pcon-RBS-luxR-DT-2(344µg/mL)EcoRISpeI10x BufferB100x BSAMilliQtotal
2 cuts2.9µL0.5µL0.5µL3.0µL0.3µL22.8µL30µL
NC0.6µl0µL0µL1µL0.1µL8.3µL10µL

Electrophoresis

Hirano

LaneSampleEnzyme1Enzyme2
100bp ladder----
1pT181 attenuatorEcoRISpeI
2pT181 attenuator----
3DT-(1)EcoRIXbaI
4DT-(1)----
5Pcon-RBS-luxR-DT-(2)EcoRISpeI
6Pcon-RBS-luxR-DT-(2)----
100bp ladder----

Igku Aug27 Electrophoresis(N2)-2.jpg

Gel Extraction

Inoue

LaneDNAEnzyme
1100bpladder--
2pT181 attenuator-1EcoRI&SpeI
3pT181 attenuator-1EcoRI&SpeI
4DT-1EcoRI&XbaI
5DT-1EcoRI&XbaI
6Pcon-RBS-luxR-DT-2EcoRI&SpeI
7Pcon-RBS-luxR-DT-2EcoRI&SpeI
8100bpladder--

Igku Aug27 Gel Extraction(N3)-1-3.jpg
Igku Aug27 Gel Extraction(N4)pic(1).jpg

Nameconcentration[µg/mL]260/280260/230
pT181 attenuator-1(EcoRI&SpeI)4.21.620.29
DT-1(EcoRI&XbaI)171.880.88

Transformation

Kojima and Hirano

NameSampleCompetent CellsTotalPlate
8/26 tetR aptamer 12_1R-DT1µL10µL11µLCP
8/26 pT181 attenuator-DT1µL10µL11µLCP
8/26 pT181 antisense-DT1µL10µL11µLCP
8/26 Spinach-DT1µL10µL11µLCP
8/26 Plac-pT181 attenuator1µL10µL11µLCP
8/26 Pcon-pT181 attenuator1µL10µL11µLAmp
8/26 Plac-pT181 antisense1µL10µL11µLCP
8/26 Pcon-pT181 antisense1µL10µL11µLAmp

Liquid culture

No name

Samplemedium
8/16 PluxPlusgrow medium(+CP)

Genome PCR

Yoshdia and Nakagawa

genome DNAKOD plus10x bufferdNTPMgSO4SasA_fwd primerSasA_rev primerMilliQtotal
0.50.52.52.51.50.750.751625
genome DNAKOD plus10x bufferdNTPMgSO4RpaA_fwd primerRpaA_rev primerMilliQtotal
0.50.52.52.51.50.750.751625
genome DNAKOD plus10x bufferdNTPMgSO4RpaB_fwd primerRpaB_rev primerMilliQtotal
0.50.52.52.51.50.750.751625
genome DNAKOD plus10x bufferdNTPMgSO4PkaiBC_fwd primerPkaiBC_rev primerMilliQtotal
0.50.52.52.51.50.750.751625
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C50°C68°C--
2 min10 sec30 sec38 sec30 cycles

Ligation

No name

stateVectorInserter
experiment8/27 DT (EcoRI & XbaI)2.88/21 RBS-lysis3 (EcoRI & SpeI)
experiment8/27 DT (EcoRI & XbaI)2.88/27 pT181 attenuator (EcoRI & SpeI)
  • Samples were evaporeted used evaporator into about 3 µL.
sampleMilliQLigation Hightotal
343.510.5
  • incubate overnight at 4 °C
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