Template:Kyoto/Notebook/Sep 22

From 2013.igem.org

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Line 1: Line 1:
==Sep 22==
==Sep 22==
 +
===Colony PCR===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Sample||base pair
 +
|-
 +
|pSB1C3+P-KaiBC①||283
 +
|-
 +
|pSB1C3+P-KaiBC②||283
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
 +
|-
 +
|2min||30s||30s||18s||30cycles
 +
|}
 +
 +
===Miniprep===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{|class="wikitable"
 +
!DNA||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|P&lambda;-luxI||225　||1.59　||0.83
 +
|}
 +
</div>
 +
 +
===Colony PCR===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Sample||base pair
 +
|-
 +
|pSB1C3+P-KaiBC①||283
 +
|-
 +
|pSB1C3+P-KaiBC②||283
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
 +
|-
 +
|2min||30s||30s||18s||30cycles
 +
|}
 +
 +
===Gel Extraction===
 +
<div class="experiment">
 +
<span class="author">Nakamoto</span>
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1||100bp ladder||--
 +
|-
 +
|3||RBS-lacZ&alpha;-DT||XbaI&PstI
 +
|-
 +
|4||RBS-lacZ&alpha;-DT||XbaI&PstI
 +
|-
 +
|5||RBS-lacZ&alpha;-DT||XbaI&PstI
 +
|-
 +
|6||RBS-lacZ&alpha;-DT||XbaI&PstI
 +
|}
 +
 +
[[File:igku_Aug19electrophoresis3]]<br>
 +
[[File:igku_Aug19electrophoresis4]]<br>
 +
{| class="wikitable"
 +
!Name||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|RBS-lacZ&alpha;-DT(XbaI&PstI)||9.8||1.50||0.39
 +
|-
 +
|}
 +
</div>
 +
 +
===digestion===
 +
 +
 +
===Gel Extraction===
 +
<div class="experiment">
 +
<span class="author">Nakamoto</span>
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1||100bp ladder||--
 +
|-
 +
|3||RBS-lacZ&alpha;-DT||XbaI&PstI
 +
|-
 +
|4||RBS-lacZ&alpha;-DT||XbaI&PstI
 +
|-
 +
|5||RBS-lacZ&alpha;-DT||XbaI&PstI
 +
|-
 +
|6||RBS-lacZ&alpha;-DT||XbaI&PstI
 +
|}
 +
 +
[[File:igku_Aug19electrophoresis3]]<br>
 +
[[File:igku_Aug19electrophoresis4]]<br>
 +
{| class="wikitable"
 +
!Name||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|RBS-lacZ&alpha;-DT(XbaI&PstI)||9.8||1.50||0.39
 +
|-
 +
|}
 +
</div>
 +
 +
 +
===Colony PCR===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Sample||base pair
 +
|-
 +
|pSB1C3+P-KaiBC①||283
 +
|-
 +
|pSB1C3+P-KaiBC②||283
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
 +
|-
 +
|2min||30s||30s||18s||30cycles
 +
|}
 +
 +
===Colony PCR===
 +
<div class="experiment">
 +
<span class="author">Tatsui</span>
 +
{| class="wikitable"
 +
!Sample||base pair
 +
|-
 +
|pSB1C3+P-KaiBC①||283
 +
|-
 +
|pSB1C3+P-KaiBC②||283
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
 +
|-
 +
|2min||30s||30s||18s||30cycles
 +
|}
 +
 +
===Colony PCR===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Sample||base pair
 +
|-
 +
|pSB1C3+P-KaiBC①||283
 +
|-
 +
|pSB1C3+P-KaiBC②||283
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
 +
|-
 +
|2min||30s||30s||18s||30cycles
 +
|}
 +
 +
===Ligation===
 +
<div class="experiment">
 +
<span class="author">Okazaki, Kanaoka, Tatsui and Hirano</span>
 +
{| class="wikitable"
 +
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
 +
|-
 +
|experiment||9/18 Plac (SpeI & PstI)||3.0 &micro;L||9/18 RBS-lysis3-DT (XbaI & PstI)||12.4 &micro;L||3.5 &micro;L
 +
|-
 +
|experiment||9/12 Ptet-pT181 antisense||1.0 &micro;L||9/18 spinach-DT||11.9 &micro;L||3.5 &micro;L
 +
|-
 +
|experiment||9/13 Pcon-RBS-luxR-DT||2.1 &micro;L||9/10 Plux-RBS-GFP-DT||5.6 &micro;L||3.5 &micro;L
 +
|-
 +
|experiment||9/13 Pcon-pT181 attenuator (SpeI & PstI)||2.0 &micro;L||9/18 aptamer 12-1R-DT (XbaI & PstI)||6.3 &micro;L||3.5 &micro;L
 +
|-
 +
|experiment||9/18 Plac &#9313;(SpeI & PstI)||3.0 &micro;L||9/18 aptamer 12-1R-DT(XbaI & PstI)||4.5&micro;L||x3.5 &micro;L
 +
|-
 +
|}
 +
</div>
 +
 +
===Liquid Culture===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Sample||medium
 +
|-
 +
|9/2 spinach-DT-2||--
 +
|-
 +
|8/16 Plux-4||--
 +
|-
 +
|8/27 Plux-RBS-GFP-DT-1||--
 +
|-
 +
|9/5 Pcon-pT181 attenuator-1||--
 +
|}
 +
</div>
 +
 +
===digestion===
 +
 +
===Gel Extraction===
 +
<div class="experiment">
 +
<span class="author">Nakamoto</span>
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1||100bp ladder||--
 +
|-
 +
|3||RBS-lacZ&alpha;-DT||XbaI&PstI
 +
|-
 +
|4||RBS-lacZ&alpha;-DT||XbaI&PstI
 +
|-
 +
|5||RBS-lacZ&alpha;-DT||XbaI&PstI
 +
|-
 +
|6||RBS-lacZ&alpha;-DT||XbaI&PstI
 +
|}
 +
 +
[[File:igku_Aug19electrophoresis3]]<br>
 +
[[File:igku_Aug19electrophoresis4]]<br>
 +
{| class="wikitable"
 +
!Name||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|RBS-lacZ&alpha;-DT(XbaI&PstI)||9.8||1.50||0.39
 +
|-
 +
|}
 +
</div>
 +
 +
 +
===Transformation===
 +
<div class="experiment">
 +
<span class="author">Tatsui</span>
 +
{| class="wikitable"
 +
!Name||Sample||Competent Cells||Plate
 +
|-
 +
|9/17 pSB1C3-Pcon-pT181 attenuator-DT||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/17 pSB1C3-Pcon-aptamer 12_1R-DT||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/17 pSB1C3-Pcon-antisense-spinach-DT||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/17 pSB1C3-Pcon-RBS-tetR-DT x2||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/14 pSB1C3-PkaiBC||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/14 pSB1C3-RpaB||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/16 pSB1C3-pT181 attenuator(EcoRI&SpeI) ||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/17 pSB1C3-pT181 attenuator(XbaI&PstI) ||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/17 pSB1C3-Pcon-spinach-DT||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/14 RBS-GFP-DT-PkaiBC||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|Plux-RBS-lysis3-DT||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/20 Ptet-pT181 antisense-spinach-DT||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/20 Pcon-spinach-DT(pSB4K5) ||2 &micro;L||20 &micro;L||Kan
 +
|-
 +
|9/20 Pcon-pT181 antisense-spinach-DT(pSB4K5) ||2 &micro;L||20 &micro;L||Kan
 +
|-
 +
|9/20 KaiABC||2 &micro;L||20 &micro;L||Kan
 +
|-
 +
|9/20 KaiABC||2 &micro;L||20 &micro;L||Kan
 +
|-
 +
|9/20 Plac-RBS-lysis3-DT||2 &micro;L||20 &micro;L||Amp
 +
|-
 +
|9/20 Plac-aptamer 12_1R||2 &micro;L||20 &micro;L||Amp
 +
|-
 +
|9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT||2 &micro;L||20 &micro;L||Amp
 +
|-
 +
|Plac-aptamer12_1R-DT||2 &micro;L||20 &micro;L||Amp
 +
|-
 +
|9/20 Plac-aptamer12_1R-DT||2 &micro;L||20 &micro;L||Amp
 +
|-
 +
|9/20Pcon-pT181attenuator-aptamer-DT||2 &micro;L||20 &micro;L||Amp
 +
|-
 +
|9/17 Plac-pT181-attenuatorx2||2 &micro;L||20 &micro;L||Amp
 +
|-
 +
|9/15 Pcon-RBS-lacZ&alpha;-DT||2 &micro;L||20 &micro;L||Amp
 +
|-
 +
|9/14 Pcon-pT181attenuator-RBS-lacZ&alpha;-DT||2 &micro;L||20 &micro;L||Amp
 +
|-
 +
|PUC19||2 &micro;L||20 &micro;L||Amp
 +
|}
 +
</div>
 +
 +
===PCR===
 +
<div class="experiment">
 +
<span class="author">Tatsui</span>
 +
{| class="wikitable"
 +
!Sample||base pair
 +
|-
 +
|pT181 attenuator(pSB1C3)||--
 +
|-
 +
|Pcon-RBS-lacZ&alpha;-DT||--
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94 &deg;C||94 &deg;C||55 &deg;C||68 &deg;C||--
 +
|-
 +
|5 min||30 s||30 s||3 min||30 cycles
 +
|}
 +
</div>
 +
 +
 +
===Transformation===
 +
<div class="experiment">
 +
<span class="author">Tatsui</span>
 +
{| class="wikitable"
 +
!Name||Sample||Competent Cells||Plate
 +
|-
 +
|9/17 pSB1C3-Pcon-pT181 attenuator-DT||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/17 pSB1C3-Pcon-aptamer 12_1R-DT||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/17 pSB1C3-Pcon-antisense-spinach-DT||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/17 pSB1C3-Pcon-RBS-tetR-DT x2||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/14 pSB1C3-PkaiBC||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/14 pSB1C3-RpaB||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/16 pSB1C3-pT181 attenuator(EcoRI&SpeI) ||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/17 pSB1C3-pT181 attenuator(XbaI&PstI) ||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/17 pSB1C3-Pcon-spinach-DT||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/14 RBS-GFP-DT-PkaiBC||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|Plux-RBS-lysis3-DT||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/20 Ptet-pT181 antisense-spinach-DT||2 &micro;L||20 &micro;L||CP
 +
|-
 +
|9/20 Pcon-spinach-DT(pSB4K5) ||2 &micro;L||20 &micro;L||Kan
 +
|-
 +
|9/20 Pcon-pT181 antisense-spinach-DT(pSB4K5) ||2 &micro;L||20 &micro;L||Kan
 +
|-
 +
|9/20 KaiABC||2 &micro;L||20 &micro;L||Kan
 +
|-
 +
|9/20 KaiABC||2 &micro;L||20 &micro;L||Kan
 +
|-
 +
|9/20 Plac-RBS-lysis3-DT||2 &micro;L||20 &micro;L||Amp
 +
|-
 +
|9/20 Plac-aptamer 12_1R||2 &micro;L||20 &micro;L||Amp
 +
|-
 +
|9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT||2 &micro;L||20 &micro;L||Amp
 +
|-
 +
|Plac-aptamer12_1R-DT||2 &micro;L||20 &micro;L||Amp
 +
|-
 +
|9/20 Plac-aptamer12_1R-DT||2 &micro;L||20 &micro;L||Amp
 +
|-
 +
|9/20Pcon-pT181attenuator-aptamer-DT||2 &micro;L||20 &micro;L||Amp
 +
|-
 +
|9/17 Plac-pT181-attenuatorx2||2 &micro;L||20 &micro;L||Amp
 +
|-
 +
|9/15 Pcon-RBS-lacZ&alpha;-DT||2 &micro;L||20 &micro;L||Amp
 +
|-
 +
|9/14 Pcon-pT181attenuator-RBS-lacZ&alpha;-DT||2 &micro;L||20 &micro;L||Amp
 +
|-
 +
|PUC19||2 &micro;L||20 &micro;L||Amp
 +
|}
 +
</div>

Revision as of 15:38, 25 September 2013

Contents

Sep 22

Colony PCR

No name

Samplebase pair
pSB1C3+P-KaiBC①283
pSB1C3+P-KaiBC②283
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
2min30s30s18s30cycles

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
Pλ-luxI225　1.59　0.83

Colony PCR

No name

Samplebase pair
pSB1C3+P-KaiBC①283
pSB1C3+P-KaiBC②283
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
2min30s30s18s30cycles

Gel Extraction

Nakamoto

LaneDNAEnzyme
1100bp ladder--
3RBS-lacZα-DTXbaI&PstI
4RBS-lacZα-DTXbaI&PstI
5RBS-lacZα-DTXbaI&PstI
6RBS-lacZα-DTXbaI&PstI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(XbaI&PstI)9.81.500.39

digestion

Gel Extraction

Nakamoto

LaneDNAEnzyme
1100bp ladder--
3RBS-lacZα-DTXbaI&PstI
4RBS-lacZα-DTXbaI&PstI
5RBS-lacZα-DTXbaI&PstI
6RBS-lacZα-DTXbaI&PstI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(XbaI&PstI)9.81.500.39


Colony PCR

No name

Samplebase pair
pSB1C3+P-KaiBC①283
pSB1C3+P-KaiBC②283
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
2min30s30s18s30cycles

Colony PCR

Tatsui

Samplebase pair
pSB1C3+P-KaiBC①283
pSB1C3+P-KaiBC②283
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
2min30s30s18s30cycles

Colony PCR

No name

Samplebase pair
pSB1C3+P-KaiBC①283
pSB1C3+P-KaiBC②283
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
2min30s30s18s30cycles

Ligation

Okazaki, Kanaoka, Tatsui and Hirano

stateVectorInserterLigation High ver.2
experiment9/18 Plac (SpeI & PstI)3.0 µL9/18 RBS-lysis3-DT (XbaI & PstI)12.4 µL3.5 µL
experiment9/12 Ptet-pT181 antisense1.0 µL9/18 spinach-DT11.9 µL3.5 µL
experiment9/13 Pcon-RBS-luxR-DT2.1 µL9/10 Plux-RBS-GFP-DT5.6 µL3.5 µL
experiment9/13 Pcon-pT181 attenuator (SpeI & PstI)2.0 µL9/18 aptamer 12-1R-DT (XbaI & PstI)6.3 µL3.5 µL
experiment9/18 Plac ②(SpeI & PstI)3.0 µL9/18 aptamer 12-1R-DT(XbaI & PstI)4.5µLx3.5 µL

Liquid Culture

No name

Samplemedium
9/2 spinach-DT-2--
8/16 Plux-4--
8/27 Plux-RBS-GFP-DT-1--
9/5 Pcon-pT181 attenuator-1--

digestion

Gel Extraction

Nakamoto

LaneDNAEnzyme
1100bp ladder--
3RBS-lacZα-DTXbaI&PstI
4RBS-lacZα-DTXbaI&PstI
5RBS-lacZα-DTXbaI&PstI
6RBS-lacZα-DTXbaI&PstI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(XbaI&PstI)9.81.500.39


Transformation

Tatsui

NameSampleCompetent CellsPlate
9/17 pSB1C3-Pcon-pT181 attenuator-DT2 µL20 µLCP
9/17 pSB1C3-Pcon-aptamer 12_1R-DT2 µL20 µLCP
9/17 pSB1C3-Pcon-antisense-spinach-DT2 µL20 µLCP
9/17 pSB1C3-Pcon-RBS-tetR-DT x22 µL20 µLCP
9/14 pSB1C3-PkaiBC2 µL20 µLCP
9/14 pSB1C3-RpaB2 µL20 µLCP
9/16 pSB1C3-pT181 attenuator(EcoRI&SpeI) 2 µL20 µLCP
9/17 pSB1C3-pT181 attenuator(XbaI&PstI) 2 µL20 µLCP
9/17 pSB1C3-Pcon-spinach-DT2 µL20 µLCP
9/14 RBS-GFP-DT-PkaiBC2 µL20 µLCP
Plux-RBS-lysis3-DT2 µL20 µLCP
9/20 Ptet-pT181 antisense-spinach-DT2 µL20 µLCP
9/20 Pcon-spinach-DT(pSB4K5) 2 µL20 µLKan
9/20 Pcon-pT181 antisense-spinach-DT(pSB4K5) 2 µL20 µLKan
9/20 KaiABC2 µL20 µLKan
9/20 KaiABC2 µL20 µLKan
9/20 Plac-RBS-lysis3-DT2 µL20 µLAmp
9/20 Plac-aptamer 12_1R2 µL20 µLAmp
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT2 µL20 µLAmp
Plac-aptamer12_1R-DT2 µL20 µLAmp
9/20 Plac-aptamer12_1R-DT2 µL20 µLAmp
9/20Pcon-pT181attenuator-aptamer-DT2 µL20 µLAmp
9/17 Plac-pT181-attenuatorx22 µL20 µLAmp
9/15 Pcon-RBS-lacZα-DT2 µL20 µLAmp
9/14 Pcon-pT181attenuator-RBS-lacZα-DT2 µL20 µLAmp
PUC192 µL20 µLAmp

PCR

Tatsui

Samplebase pair
pT181 attenuator(pSB1C3)--
Pcon-RBS-lacZα-DT--
PreDenatureDenatureAnnealingExtensioncycle
94 °C94 °C55 °C68 °C--
5 min30 s30 s3 min30 cycles


Transformation

Tatsui

NameSampleCompetent CellsPlate
9/17 pSB1C3-Pcon-pT181 attenuator-DT2 µL20 µLCP
9/17 pSB1C3-Pcon-aptamer 12_1R-DT2 µL20 µLCP
9/17 pSB1C3-Pcon-antisense-spinach-DT2 µL20 µLCP
9/17 pSB1C3-Pcon-RBS-tetR-DT x22 µL20 µLCP
9/14 pSB1C3-PkaiBC2 µL20 µLCP
9/14 pSB1C3-RpaB2 µL20 µLCP
9/16 pSB1C3-pT181 attenuator(EcoRI&SpeI) 2 µL20 µLCP
9/17 pSB1C3-pT181 attenuator(XbaI&PstI) 2 µL20 µLCP
9/17 pSB1C3-Pcon-spinach-DT2 µL20 µLCP
9/14 RBS-GFP-DT-PkaiBC2 µL20 µLCP
Plux-RBS-lysis3-DT2 µL20 µLCP
9/20 Ptet-pT181 antisense-spinach-DT2 µL20 µLCP
9/20 Pcon-spinach-DT(pSB4K5) 2 µL20 µLKan
9/20 Pcon-pT181 antisense-spinach-DT(pSB4K5) 2 µL20 µLKan
9/20 KaiABC2 µL20 µLKan
9/20 KaiABC2 µL20 µLKan
9/20 Plac-RBS-lysis3-DT2 µL20 µLAmp
9/20 Plac-aptamer 12_1R2 µL20 µLAmp
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT2 µL20 µLAmp
Plac-aptamer12_1R-DT2 µL20 µLAmp
9/20 Plac-aptamer12_1R-DT2 µL20 µLAmp
9/20Pcon-pT181attenuator-aptamer-DT2 µL20 µLAmp
9/17 Plac-pT181-attenuatorx22 µL20 µLAmp
9/15 Pcon-RBS-lacZα-DT2 µL20 µLAmp
9/14 Pcon-pT181attenuator-RBS-lacZα-DT2 µL20 µLAmp
PUC192 µL20 µLAmp
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