Template:Kyoto/Notebook/Aug 28

From 2013.igem.org

(Difference between revisions)
(→Colony PCR)
(→Liquid Culture)
 
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Line 20: Line 20:
|7||100bp
|7||100bp
|}
|}
-
[[File:igku_xxxxxx.xxx]]<br>
+
[[File:Igku Aug28 Electrophoresis(N①-1).jpg]]<br>
</div>
</div>
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!state||colspan="2"|Vector||colspan="2"|Inserter
!state||colspan="2"|Vector||colspan="2"|Inserter
|-
|-
-
|experiment||8/71 DT-1||3.0||8/20 RBS-lysis3-1|8.0
+
|experiment||8/17 DT-1||3.0||8/20 RBS-lysis3-1|8.0
|}
|}
*Samples were evaporeted used evaporator into about 3 &micro;L.
*Samples were evaporeted used evaporator into about 3 &micro;L.
Line 67: Line 67:
|9||PkaiBC(8/27 Genome PCR production)
|9||PkaiBC(8/27 Genome PCR production)
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:Igku Aug28 Gel Extraction(N②pic)before.jpg]]<br>
-
[[File:igku_xxafterxx.xxx]]<br>
+
[[File:Igku Aug28 Gel Extraction(N①pic)after.jpg]]<br>
 +
 
 +
{| class="wikitable"
 +
!Name||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|RpaA||15.4||1.88||0.03
 +
|-
 +
|SasA||21.6||1.66||0.79
 +
|-
 +
|PkaiBC||16.3||1.79||0.68
 +
|-
 +
|RpaB||26.7||1.77||0.82
 +
|}
===Colony PCR===
===Colony PCR===
Line 91: Line 103:
|5min||30s||30s||48s||30cycles
|5min||30s||30s||48s||30cycles
|}
|}
-
[[File:igku_Aug19electrophoresis1.png]]
+
[[File:Igku Aug28 Colony PCR(N③pic).jpg]]
</div>
</div>
Line 159: Line 171:
|4||8/17 RBS-GFP-DT-(2)||--||--
|4||8/17 RBS-GFP-DT-(2)||--||--
|-
|-
-
|5||8/20 Pcon-RBS-GFP-DT-(1)||EcoRI||XbaI
+
|5||8/20 Pcon-RBS-GFP-DT-(1)||EcoRI||SpeI
|-
|-
|6||8/20 Pcon-RBS-GFP-DT-(1)||--||--
|6||8/20 Pcon-RBS-GFP-DT-(1)||--||--
Line 177: Line 189:
|13||8/20 RBS-tetR-DT||--||--
|13||8/20 RBS-tetR-DT||--||--
|}
|}
-
[[File:igku_Aug28electrophoresis1]]<br>
+
[[File:Igku Aug28 Electrophoresis(ColoP)(N4)pic.jpg]]<br>
</div>
</div>
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|6
|6
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:Igku Aug28 Gel Extraction(N⑤-1,2)before.jpg]]<br>
-
[[File:igku_xxafterxx.xxx]]<br>
+
[[File:Igku Aug28 Gel Extraction4-2.jpg]]<br>
{| class="wikitable"
{| class="wikitable"
!Lane||DNA||Enzyme
!Lane||DNA||Enzyme
Line 215: Line 227:
|6
|6
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:Igku Aug28 Gel Extraction(N⑤-3).jpg]]<br>
-
[[File:igku_xxafterxx.xxx]]<br>
+
{| class="wikitable"
{| class="wikitable"
!Name||concentration[&micro;g/mL]||260/280||260/230
!Name||concentration[&micro;g/mL]||260/280||260/230
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|5min||30s||30s||30s||30cycles
|5min||30s||30s||30s||30cycles
|}
|}
-
[[File:igku_Aug28electrophoresis3?.png]]
+
[[File:Igku Aug28 c(N⑤-1,2)before.jpg]]
{| class="wikitable"
{| class="wikitable"
!|Sample||base pair
!|Sample||base pair
|-
|-
-
|8/27 Pλ-luxI-1||???
+
|8/27 P&lambda;-luxI-1||--
|-
|-
-
|8/27 Pλ-luxI||???
+
|8/27 P&lambda;-luxI||--
|}
|}
{| class="wikitable"
{| class="wikitable"
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|5min||30s||30s||30min||3cycles
|5min||30s||30s||30min||3cycles
|}
|}
-
[[File:igku_Aug19electrophoresis1.png]]
+
[[File:Igku Aug28 ColonyPCR(N⑦-2).jpg]]
</div>
</div>
 +
 +
===Colony PCR===
 +
<div class="expeiment">
 +
<span class>No name</span>
 +
{| class="wikitable"
 +
!|Sample||base pair
 +
|-
 +
|8/21 RBS-lysis2-9||772
 +
|-
 +
|8/21 RBS-lysis2-10||772
 +
|-
 +
|8/21 RBS-lysis2-11||772
 +
|-
 +
|8/21 RBS-lysis2-12||772
 +
|-
 +
|8/21 RBS-lysis2-13||772
 +
|-
 +
|8/21 RBS-lysis2-14||772
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
 +
|-
 +
|5min||30s||30s||48s||30cycles
 +
|}
===Liquid Culture===
===Liquid Culture===
Line 283: Line 320:
|8/16 Master Plate(CP)-6 DT||Plusgrow medium(+CP)
|8/16 Master Plate(CP)-6 DT||Plusgrow medium(+CP)
|-
|-
-
|J231Master Plate-1||Plusgrow medium(+Amp)
+
|J23100 Master Plate-1||Plusgrow medium(+Amp)
|-
|-
-
|Plac Master Plate-1||Plusgrow medium(+CP)　
+
|Plac Master Plate-1||Plusgrow medium(+CP)
|}
|}
</div>
</div>
Line 297: Line 334:
|experiment||8/27 DT (EcoRI & XbaI)||2.9||8/28 RBS-lysis1 (EcoRI & SpeI)||9.5
|experiment||8/27 DT (EcoRI & XbaI)||2.9||8/28 RBS-lysis1 (EcoRI & SpeI)||9.5
|-
|-
-
|experiment||8/28 Plux (SpeI & PstI)||2.3||8/28 RBS-GFP-DT (XbaI & PstI)||
+
|experiment||8/28 Plux (SpeI & PstI)||2.3||8/28 RBS-GFP-DT (XbaI & PstI)||18
|-
|-
-
|experiment||8/28 Pcon-RBS-luxR-DT (EcoRI&XbaI)||3.3||8/28 Pcon^RBS-GFP-DT-(1) (EcoRI&SpeI)||28
+
|experiment||8/28 Pcon-RBS-luxR-DT (EcoRI & XbaI)||3.3||8/28 Pcon^RBS-GFP-DT-(1) (EcoRI & SpeI)||28
|-
|-
-
|8/27 DT (EcoRI&XbaI)||2.7||8/24 Spinach (EcoRI&SpeI)||2.4
+
|experiment||8/27 DT (EcoRI & XbaI)||2.7||8/24 Spinach (EcoRI & SpeI)||2.4
|}
|}
*Samples were evaporeted used evaporator into about 1 &micro;L.
*Samples were evaporeted used evaporator into about 1 &micro;L.
Line 318: Line 355:
!Sample||medium
!Sample||medium
|-
|-
-
|8/26 Pλ-RBS-luxI-DT-1||Plusgrow medium (+Amp)  
+
|8/26 P&lambda;-RBS-luxI-DT-1||Plusgrow medium (+Amp)  
|-
|-
-
|8/26 Pλ-RBS-luxI-DT-2||Plusgrow medium (+Amp)　
+
|8/26 P&lambda;-RBS-luxI-DT-2||Plusgrow medium (+Amp)
|}
|}
* incubate 37&deg;C 10hour
* incubate 37&deg;C 10hour
</div>
</div>

Latest revision as of 03:46, 28 September 2013

Contents

Aug 28

Electrophoresis

No name

LaneSample
1100bp
2SasA
3RpaA
4RpaB
5PkaiBC
6PCR NC
7100bp

Igku Aug28 Electrophoresis(N①-1).jpg

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
8/27 Plux173.81.951.45

Ligation

No name

stateVectorInserter
experiment8/17 DT-13.08.0
  • Samples were evaporeted used evaporator into about 3 µL.
sampleMilliQLigation Hightotal
343.510.5
  • incubate one hour at 16 °C

Gel Extraction

No name

LaneDNA
1100bpladder
3SasA(8/27 Genome PCR production)
5RpaA(8/27 Genome PCR production)
7RpaB(8/27 Genome PCR production)
9PkaiBC(8/27 Genome PCR production)

Igku Aug28 Gel Extraction(N②pic)before.jpg
Igku Aug28 Gel Extraction(N①pic)after.jpg

Nameconcentration[µg/mL]260/280260/230
RpaA15.41.880.03
SasA21.61.660.79
PkaiBC16.31.790.68
RpaB26.71.770.82

Colony PCR

No name

Samplebase pair
8/21 RBS-lysis2-(3)772
8/21 RBS-lysis2-(4)772
8/21 RBS-lysis2-(5)772
8/21 RBS-lysis2-(6)772
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s48s30cycles

Igku Aug28 Colony PCR(N③pic).jpg

Restriction Enzyme Digestion

No name

8/22 RBS-lysis1-(1)EcoRISpeIBufferBBSAMilliQtotal
2 cuts9µL0.5µL0.5µL3µL0.3µL16.7µL30µL
NC1µL0µL0µL1µL0.1µL7.9µL10µL
8/17 RBS-GFP-DT-(2)XbaIPstIBufferDBSAMilliQtotal
2 cuts4µL0.5µL0.5µL3µL0.3µL21.7µL30µL
NC0.5µL0µL0µL1µL0.1µL8.4µL10µL
8/20 Pcon-RBS-GFP-DT-(1)EcoRISpeIBufferBBSAMilliQtotal
2 cuts3µL0.5µL0.5µL3µL0.3µL22.7µL30µL
NC0.3µL0µL0µL1µL0.1µL8.6µL10µL
8/20 Pcon-RBS-luxR-DT-(1)EcoRIXbaIBufferDBSAMilliQtotal
2 cuts2µL0.5µL0.5µL3µL0.3µL23.7µL30µL
NC0.3µL0µL0µL1µL0.1µL8.6µL10µL

No name

8/28 PluxSpeIPstIBufferDBSAMilliQtotal
2 cuts6µL0.5µL0.5µL3µL0.3µL19.7µL30µL
NC1µL0µL0µL1µL0.1µL7.9µL10µL
8/20 RBS-tetR-DT-(1)XbaIPstIBufferDBSAMilliQtotal
2 cuts5µL0.5µL0.5µL3µL0.3µL20.7µL30µL
NC0.5µL0µL0µL1µL0.1µL8.4µL10µL
  • incubated 37°C 1hour

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
18/22 RBS-lysis1-(1)EcoRISpeI
28/22 RBS-lysis1-(1)----
38/17 RBS-GFP-DT-(2)XbaIPstI
48/17 RBS-GFP-DT-(2)----
58/20 Pcon-RBS-GFP-DT-(1)EcoRISpeI
68/20 Pcon-RBS-GFP-DT-(1)----
7100bp ladder----
88/20 Pcon-RBS-luxR-DT-(1)EcoRIXbaI
98/20 Pcon-RBS-luxR-DT-(1)----
108/28 PluxSpeIPstI
118/28 Plux----
128/20 RBS-tetR-DTXbaIPstI
138/20 RBS-tetR-DT----

Igku Aug28 Electrophoresis(ColoP)(N4)pic.jpg

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
28/20 RBS-lysis1-(1)EcoRI & SpeI
3
4----
58/28 RBS-GFP-DT-(2)XbaI & PstI
6

Igku Aug28 Gel Extraction(N⑤-1,2)before.jpg
Igku Aug28 Gel Extraction4-2.jpg

LaneDNAEnzyme
1100bp ladder--
28/28 PluxSpeI & PstI
3
4----
58/20 RBS-tetR-DT-(1)XbaI & PstI
6

Igku Aug28 Gel Extraction(N⑤-3).jpg

Nameconcentration[µg/mL]260/280260/230
Pcon-RBS-GFP-DT (EcoRI&SpeI)7.11.880.07
Pcon-RBS-luxR-DT (EcoRI&XbaI)15.31.930.70
Plux (SpeI&PstI)4.51.790.31
RBS-GFP-DT (XbaI&PstI)6.02.160.03
RBS-tetR-DT (XbaI&PstI)7.61.910.38

Colony PCR

No name

Samplebase pair
8/27 pT181 attenuator-DT-1738
8/27 pT181 attenuator-DT-2738
8/27 pT181 attenuator-DT-3738
8/27 TetR-aptamer 12_1R-DT-1521
8/27 TetR-aptamer 12_1R-DT-2521
8/27 pT181 antisense-DT-1542
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30s30cycles

Igku Aug28 c(N⑤-1,2)before.jpg

Samplebase pair
8/27 Pλ-luxI-1--
8/27 Pλ-luxI--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30min3cycles

Igku Aug28 ColonyPCR(N⑦-2).jpg

Colony PCR

No name

Samplebase pair
8/21 RBS-lysis2-9772
8/21 RBS-lysis2-10772
8/21 RBS-lysis2-11772
8/21 RBS-lysis2-12772
8/21 RBS-lysis2-13772
8/21 RBS-lysis2-14772
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s48s30cycles

Liquid Culture

No name

Samplemedium
8/16 Master Plate(CP)-6 DTPlusgrow medium(+CP)
J23100 Master Plate-1Plusgrow medium(+Amp)
Plac Master Plate-1Plusgrow medium(+CP)

Ligation

No name

stateVectorInserter
experiment8/27 DT (EcoRI & XbaI)2.98/28 RBS-lysis1 (EcoRI & SpeI)9.5
experiment8/28 Plux (SpeI & PstI)2.38/28 RBS-GFP-DT (XbaI & PstI)18
experiment8/28 Pcon-RBS-luxR-DT (EcoRI & XbaI)3.38/28 Pcon^RBS-GFP-DT-(1) (EcoRI & SpeI)28
experiment8/27 DT (EcoRI & XbaI)2.78/24 Spinach (EcoRI & SpeI)2.4
  • Samples were evaporeted used evaporator into about 1 µL.
sampleMilliQLigation Hightotal
1349
  • incubate overnight at 4 °C

Liquid Culture

No name

Samplemedium
8/26 Pλ-RBS-luxI-DT-1Plusgrow medium (+Amp)
8/26 Pλ-RBS-luxI-DT-2Plusgrow medium (+Amp)
  • incubate 37°C 10hour
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