Template:Kyoto/Notebook/Sep 23

From 2013.igem.org

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(→Ligation)
(→Sep 23)
 
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===Gel Extraction===
===Gel Extraction===
-
<!-- こっから -->
+
 
<div class="experiment">
<div class="experiment">
<span class="author">No Name</span>
<span class="author">No Name</span>
Line 30: Line 30:
!Name||concentration[&micro;g/mL]||260/280||260/230
!Name||concentration[&micro;g/mL]||260/280||260/230
|-
|-
-
|Pcon-PT181 anti E+S||46.7 ||1.06 ||0.93
+
|Pcon-PT181 anti EcoRI+SpeI||46.7 ||1.06 ||0.93
|-
|-
-
|Pcon-RBS-tetR-DT E+X||119.3 ||0.99 ||
+
|Pcon-RBS-tetR-DT EcoRI+XbaI||119.3 ||0.99 ||
|-
|-
-
|RBS-8FP-DT E+X||87.7 ||1.00 ||0.98
+
|RBS-8FP-DT EcoRI+XbaI||87.7 ||1.00 ||0.98
|-
|-
-
|ap42_1R-DT E+X||68.8 ||1.32 ||1.03
+
|ap42_1R-DT EcoRI+XbaI||68.8 ||1.32 ||1.03
|-
|-
|Pcon-RBS-tetR-DT 1-2 A||203.0 ||1.66 ||1.79
|Pcon-RBS-tetR-DT 1-2 A||203.0 ||1.66 ||1.79
Line 49: Line 49:
|}
|}
</div>
</div>
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<!-- ここまでをコピーしてね -->
+
 
===PCR===
===PCR===
Line 67: Line 67:
!PreDenature||Denature||Annealing||Extension||cycle
!PreDenature||Denature||Annealing||Extension||cycle
|-
|-
-
|94　&deg;C||98　&deg;C||57　&deg;C||68　&deg;C||--
+
|94 &deg;C||98 &deg;C||57 &deg;C||68 &deg;C||--
|-
|-
-
|2min　||10sec　||30sec　||20sec　||30cycles
+
|2min ||10sec ||30sec ||20sec ||30cycles
|}
|}
</div>
</div>
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<!-- ここまでをコピーしてね -->
+
 
===Ligation===
===Ligation===
   
   
-
<!-- こっから -->
+
 
<div class="experiment">
<div class="experiment">
<span class="author">Yoshida</span>
<span class="author">Yoshida</span>
Line 81: Line 81:
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
|-
|-
-
|experiment||9/21 Pcon-RBS-GFP-DT E+X||47.5 &micro;g/mL||9/15 Pcon-RBS-tetR-DT E+S||59 &micro;g/mL||2.3 &micro;g/mL
+
|experiment||9/21 Pcon-RBS-GFP-DT EcoRI+XbaI||47.5 &micro;g/mL||9/15 Pcon-RBS-tetR-DT EcoRI+SpeI||59 &micro;g/mL||2.3 &micro;g/mL
|-
|-
-
|experiment||9/21 Pcon-RBS-GFP-DT E+X||47.5 &micro;g/mL||9/15 Pcon-apt12_1R-DT E+S||17 &micro;g/mL||2.4 &micro;g/mL
+
|experiment||9/21 Pcon-RBS-GFP-DT EcoRI+XbaI||47.5 &micro;g/mL||9/15 Pcon-apt12_1R-DT EcoRI+SpeI||17 &micro;g/mL||2.4 &micro;g/mL
|-
|-
-
|experiment||9/21 Pcon-RBS-GFRDT E+X||2.1 &micro;g/mL||9/15 Pcon-DT181 attenuator-DT||21 &micro;g/mL||3.0 &micro;g/mL
+
|experiment||9/21 Pcon-RBS-GFRDT EcoRI+XbaI||2.1 &micro;g/mL||9/15 Pcon-DT181 attenuator-DT||21 &micro;g/mL||3.0 &micro;g/mL
|-
|-
-
|experiment||9/22 apt 12_1R-DT E+X||67.8 &micro;g/mL||9/18 Pcon-DT181 atte E+S||5.7 &micro;g/mL||3.5 &micro;g/mL
+
|experiment||9/22 apt 12_1R-DT EcoRI+XbaI||67.8 &micro;g/mL||9/18 Pcon-DT181 attenuator EcoRI+SpeI||5.7 &micro;g/mL||3.5 &micro;g/mL
|-
|-
-
|experiment||9/22RBS-GFP-DT E+X||87.7 &micro;g/mL||9/18 Pcon-DT18 atte E+S||6.7 &micro;g/mL||3.5 &micro;g/mL
+
|experiment||9/22RBS-GFP-DT EcoRI+XbaI||87.7 &micro;g/mL||9/18 Pcon-DT18 attenuator EcoRI+SpeI||6.7 &micro;g/mL||3.5 &micro;g/mL
|-
|-
-
|experiment||9/13 DT E+X||25.4 &micro;g/mL||9/22 Pcon-DT181 anti E+S||46.8 &micro;g/mL||2.3 &micro;g/mL
+
|experiment||9/13 DT EcoRI+XbaI||25.4 &micro;g/mL||9/22 Pcon-DT181 antisense EcoRI+SpeI||46.8 &micro;g/mL||2.3 &micro;g/mL
|}
|}
</div>
</div>
-
<!-- ここまでをコピーしてね -->
+
 
===PCR===
===PCR===
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!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||SasA_fwd primer||SasA_rev primer||MilliQ||total
!genome DNA||KOD plus||10x buffer||dNTP||MgSO4||SasA_fwd primer||SasA_rev primer||MilliQ||total
|-
|-
-
|Pcon-atte-DT (E-1A) ||0.5||2.5||2.5||1.5||0.75||0.75||77.5 ||25
+
|Pcon-attenuator-DT (E-1A) ||0.5||2.5||2.5||1.5||0.75||0.75||77.5 ||25
|-
|-
-
|Pcon-atte-DT (2-1A) ||0.5||2.5||2.5||1.5||0.75||0.75||77.5 ||25
+
|Pcon-attenuator-DT (2-1A) ||0.5||2.5||2.5||1.5||0.75||0.75||77.5 ||25
|-
|-
-
|Pcon-anti-spinach-DT (1-2A) ||0.5||2.5||2.5||1.5||0.75||0.75||77.5 ||25
+
|Pcon-antisense-spinach-DT (1-2A) ||0.5||2.5||2.5||1.5||0.75||0.75||77.5 ||25
|-
|-
-
|Pcon-anti-spinach-DT (E-1A) ||0.5||2.5||2.5||1.5||0.75||0.75||77.5 ||25
+
|Pcon-antisense-spinach-DT (E-1A) ||0.5||2.5||2.5||1.5||0.75||0.75||77.5 ||25
|}
|}
{| class="wikitable"
{| class="wikitable"
!PreDenature||Denature||Annealing||Extension||cycle
!PreDenature||Denature||Annealing||Extension||cycle
|-
|-
-
|94　&deg;C||98　&deg;C||57　&deg;C||68　&deg;C||--
+
|94 &deg;C||98 &deg;C||57 &deg;C||68 &deg;C||--
|-
|-
-
|2min　||10s　||30s　||45s　||30cycle
+
|2min ||10s ||30s ||45s ||30cycle
|}
|}
</div>
</div>
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<!-- ここまでをコピーしてね -->
+
 
===Miniprep===
===Miniprep===
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!DNA||concentration[&micro;g/mL]||260/280||260/230  
!DNA||concentration[&micro;g/mL]||260/280||260/230  
|-
|-
-
|9/22 Plac-DT181 atte-1||204.0　||2.00　||1.95
+
|9/22 Plac-DT181 attenuator-1||204.0 ||2.00 ||1.95
|-
|-
-
|9/22 Plac-DT181 atte-2||195.3　||1.84　||1.62
+
|9/22 Plac-DT181 attenuator-2||195.3 ||1.84 ||1.62
|-
|-
-
|9/22 Plac-DT181 anti-1||188.2　||1.87　||2.15
+
|9/22 Plac-DT181 antisense-1||188.2 ||1.87 ||2.15
|-
|-
-
|9/22 Plac-DT181 anti-2||165.0　||1.95　||2.66
+
|9/22 Plac-DT181 antisense-2||165.0 ||1.95 ||2.66
|}
|}
</div>
</div>
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<!-- ここまでをコピーしてね -->
+
 
===Electrophoresis===
===Electrophoresis===
   
   
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|1,6||100bp ladder(Promega)||--||--
|1,6||100bp ladder(Promega)||--||--
|-
|-
-
|2||Pcon-atte-DT (E-1A)||--||--
+
|2||Pcon-attenuator-DT (E-1A)||--||--
|-
|-
-
|3||Pcon-atte-DT (2-1A)||--||--  
+
|3||Pcon-attenuator-DT (2-1A)||--||--  
|-
|-
-
|4||Pcon-anti-spinach-DT (1-2A)||--||--
+
|4||Pcon-antisense-spinach-DT (1-2A)||--||--
|-
|-
-
|5||Pcon-anti-spinach-DT (E-1A)||--||--
+
|5||Pcon-antisense-spinach-DT (E-1A)||--||--
|}
|}
[[File:igku_9232.png]]<br>
[[File:igku_9232.png]]<br>
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|1||100bp ladder||--||--   
|1||100bp ladder||--||--   
|-
|-
-
|2||9/8 Pcon-pT181 anti(E-1A)||--||--  
+
|2||9/8 Pcon-pT181 antisense(E-1A)||--||--  
|-
|-
|3||8/20 Ptet(1)(2-S)||--||--
|3||8/20 Ptet(1)(2-S)||--||--
Line 190: Line 190:
===Transformation===
===Transformation===
-
<!-- こっから -->
+
 
<div class="experiment">
<div class="experiment">
<span class="author">No Name</span>
<span class="author">No Name</span>
Line 200: Line 200:
|}
|}
</div>
</div>
-
<!-- ここまでをコピーしてね -->
+
 
===Ligation===
===Ligation===
   
   
-
<!-- こっから -->
+
 
<div class="experiment">
<div class="experiment">
<span class="author">No Name</span>
<span class="author">No Name</span>
Line 210: Line 210:
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
!state||colspan="2"|Vector||colspan="2"|Inserter||Ligation High ver.2
|-
|-
-
|experiment||9/19 Plac S+P||24.6 &micro;g/mL||9/18 ape12_1R-DT X+P||4.5 &micro;g/mL||2 &micro;g/mL
+
|experiment||9/19 Plac SpeI+PstI||24.6 &micro;g/mL||9/18 ape12_1R-DT XbaI+PstI||4.5 &micro;g/mL||2 &micro;g/mL
|-
|-
-
|experiment||9/21 RBS-GFP-DT E+X||88.4 &micro;g/mL||9/15 Pcon-pT181 attenuator E+S||21 &micro;g/mL||2 &micro;g/mL
+
|experiment||9/21 RBS-GFP-DT EcoRI+XbaI||88.4 &micro;g/mL||9/15 Pcon-pT181 attenuator EcoRI+SpeI||21 &micro;g/mL||2 &micro;g/mL
|-
|-
-
|experiment||9/13 PSB1C3 X+P||26.0 &micro;g/mL||9/16 pT181 attenuator X+P||8.2 &micro;g/mL||2 &micro;g/mL
+
|experiment||9/13 PSB1C3 XbaI+PstI||26.0 &micro;g/mL||9/16 pT181 attenuator XbaI+PstI||8.2 &micro;g/mL||2 &micro;g/mL
|}
|}
</div>
</div>
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<!-- ここまでをコピーしてね -->
+
 
===Colony PCR===
===Colony PCR===

Latest revision as of 06:51, 27 September 2013

Contents

Sep 23

Transformation

No name

NameSampleCompetent CellsPlate
9/14 pSB1C3＋PkaiBC2 µL20 µL--
9/14 pSB1C3＋RpaB2 µL20 µL--
9/14 pkaiBC＋Rbs-GFP-DT2 µL20 µL--

Gel Extraction

No Name

LaneDNAEnzyme
1 1kbp ladder
3-4 Pcon-RBS-tetR-PT

Igku Sep23 Gel Extraction1.jpg
Igku Sep23 Gel Extraction2.jpg

Nameconcentration[µg/mL]260/280260/230
Pcon-PT181 anti EcoRI+SpeI46.7 1.06 0.93
Pcon-RBS-tetR-DT EcoRI+XbaI119.3 0.99
RBS-8FP-DT EcoRI+XbaI87.7 1.00 0.98
ap42_1R-DT EcoRI+XbaI68.8 1.32 1.03
Pcon-RBS-tetR-DT 1-2 A203.0 1.66 1.79
Pcon-RBS-tetR-DT 3-2 A67.7 2.64 1.28
Pcon-RBS-GFP-DT 1-S A185.6 1.88 2.04
Pcon-RBS-GFP-DT 2-S A298.6 1.68 1.55
Pcon-tetR-DT E-2 A202.4 1.57 1.66


PCR

No name

genome DNAKOD plus10x bufferdNTPMgSO4SasA_fwd primerSasA_rev primerMilliQtotal
9/8 Pcon-pT181 anti 0.52.52.51.50.750.7515.5 25
8/20 Ptet(1) 0.52.52.51.50.750.7515.5 25
9/16 Plac 2 0.52.52.51.50.750.7515.5 25
PreDenatureDenatureAnnealingExtensioncycle
94 °C98 °C57 °C68 °C--
2min 10sec 30sec 20sec 30cycles

Ligation

Yoshida

stateVectorInserterLigation High ver.2
experiment9/21 Pcon-RBS-GFP-DT EcoRI+XbaI47.5 µg/mL9/15 Pcon-RBS-tetR-DT EcoRI+SpeI59 µg/mL2.3 µg/mL
experiment9/21 Pcon-RBS-GFP-DT EcoRI+XbaI47.5 µg/mL9/15 Pcon-apt12_1R-DT EcoRI+SpeI17 µg/mL2.4 µg/mL
experiment9/21 Pcon-RBS-GFRDT EcoRI+XbaI2.1 µg/mL9/15 Pcon-DT181 attenuator-DT21 µg/mL3.0 µg/mL
experiment9/22 apt 12_1R-DT EcoRI+XbaI67.8 µg/mL9/18 Pcon-DT181 attenuator EcoRI+SpeI5.7 µg/mL3.5 µg/mL
experiment9/22RBS-GFP-DT EcoRI+XbaI87.7 µg/mL9/18 Pcon-DT18 attenuator EcoRI+SpeI6.7 µg/mL3.5 µg/mL
experiment9/13 DT EcoRI+XbaI25.4 µg/mL9/22 Pcon-DT181 antisense EcoRI+SpeI46.8 µg/mL2.3 µg/mL


PCR

No name

genome DNAKOD plus10x bufferdNTPMgSO4SasA_fwd primerSasA_rev primerMilliQtotal
Pcon-attenuator-DT (E-1A) 0.52.52.51.50.750.7577.5 25
Pcon-attenuator-DT (2-1A) 0.52.52.51.50.750.7577.5 25
Pcon-antisense-spinach-DT (1-2A) 0.52.52.51.50.750.7577.5 25
Pcon-antisense-spinach-DT (E-1A) 0.52.52.51.50.750.7577.5 25
PreDenatureDenatureAnnealingExtensioncycle
94 °C98 °C57 °C68 °C--
2min 10s 30s 45s 30cycle


Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
9/22 Plac-DT181 attenuator-1204.0 2.00 1.95
9/22 Plac-DT181 attenuator-2195.3 1.84 1.62
9/22 Plac-DT181 antisense-1188.2 1.87 2.15
9/22 Plac-DT181 antisense-2165.0 1.95 2.66

Electrophoresis

Hirano

LaneSampleEnzyme1Enzyme2
1,6100bp ladder(Promega)----
2Pcon-attenuator-DT (E-1A)----
3Pcon-attenuator-DT (2-1A)----
4Pcon-antisense-spinach-DT (1-2A)----
5Pcon-antisense-spinach-DT (E-1A)----

Igku 9232.png

Gel Extraction

Hirano

Nameconcentration[µg/mL]260/280260/230
Pcon-pT181 attenuator-DT(E-1A)166.7 1.51 0.99
Pcon-pT181 attenuator-DT(2-1A)122.1 1.75 1.20
Pcon-pT181 antisense-spinach-DT(1-2A)99.1 1.86 1.81

Electrophoresis

Hirano

LaneSampleEnzyme1Enzyme2
1100bp ladder----
29/8 Pcon-pT181 antisense(E-1A)----
38/20 Ptet(1)(2-S)----
49/16 Plac2(E-1A)----

Igku 9231.png

Transformation

No Name

NameSampleCompetent CellsTotalPlate
pSB1C32µL20µL22µL


Ligation

No Name

stateVectorInserterLigation High ver.2
experiment9/19 Plac SpeI+PstI24.6 µg/mL9/18 ape12_1R-DT XbaI+PstI4.5 µg/mL2 µg/mL
experiment9/21 RBS-GFP-DT EcoRI+XbaI88.4 µg/mL9/15 Pcon-pT181 attenuator EcoRI+SpeI21 µg/mL2 µg/mL
experiment9/13 PSB1C3 XbaI+PstI26.0 µg/mL9/16 pT181 attenuator XbaI+PstI8.2 µg/mL2 µg/mL


Colony PCR

Tatsui

Samplebase pair
9/22 Pcon-PT181 attenuator12-1R-DT(1~8)--
9/22 Pcon-PT181 attenuator(1~4)--
9/20 Plac＋aptamer12-1R-DT(1~4)--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s54s30cycle
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