Template:Kyoto/Notebook/Aug 28

From 2013.igem.org

(Difference between revisions)
(→Miniprep)
(→Liquid Culture)
 
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|7||100bp
|7||100bp
|}
|}
-
[[File:igku_xxxxxx.xxx]]<br>
+
[[File:Igku Aug28 Electrophoresis(N①-1).jpg]]<br>
</div>
</div>
Line 34: Line 34:
===Ligation===
===Ligation===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!state||colspan="2"|Vector||colspan="2"|Inserter
 +
|-
 +
|experiment||8/17 DT-1||3.0||8/20 RBS-lysis3-1|8.0
 +
|}
 +
*Samples were evaporeted used evaporator into about 3 &micro;L.
 +
{| class="wikitable"
 +
!sample||MilliQ||Ligation High||total
 +
|-
 +
|3||4||3.5||10.5
 +
|}
 +
*incubate one hour at 16 &deg;C
 +
</div>
 +
===Gel Extraction===
===Gel Extraction===
 +
<!-- こっから -->
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Lane||DNA
 +
|-
 +
|1||100bpladder
 +
|-
 +
|3||SasA(8/27 Genome PCR production)
 +
|-
 +
|5||RpaA(8/27 Genome PCR production)
 +
|-
 +
|7||RpaB(8/27 Genome PCR production)
 +
|-
 +
|9||PkaiBC(8/27 Genome PCR production)
 +
|}
 +
[[File:Igku Aug28 Gel Extraction(N②pic)before.jpg]]<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===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!|Sample||base 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
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
 +
|-
 +
|5min||30s||30s||48s||30cycles
 +
|}
 +
[[File:Igku Aug28 Colony PCR(N③pic).jpg]]
 +
</div>
 +
===Restriction Enzyme Digestion===
===Restriction Enzyme Digestion===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
! ||8/22 RBS-lysis1-(1)||EcoRI||SpeI||BufferB||BSA||MilliQ||total
 +
|-
 +
|2 cuts||9&micro;L||0.5&micro;L||0.5&micro;L||3&micro;L||0.3&micro;L||16.7&micro;L||30&micro;L
 +
|-
 +
|NC||1&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||7.9&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
! ||8/17 RBS-GFP-DT-(2)||XbaI||PstI||BufferD||BSA||MilliQ||total
 +
|-
 +
|2 cuts||4&micro;L||0.5&micro;L||0.5&micro;L||3&micro;L||0.3&micro;L||21.7&micro;L||30&micro;L
 +
|-
 +
|NC||0.5&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.4&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
! ||8/20 Pcon-RBS-GFP-DT-(1)||EcoRI||SpeI||BufferB||BSA||MilliQ||total
 +
|-
 +
|2 cuts||3&micro;L||0.5&micro;L||0.5&micro;L||3&micro;L||0.3&micro;L||22.7&micro;L||30&micro;L
 +
|-
 +
|NC||0.3&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.6&micro;L||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
! ||8/20 Pcon-RBS-luxR-DT-(1)||EcoRI||XbaI||BufferD||BSA||MilliQ||total
 +
|-
 +
|2 cuts||2&micro;L||0.5&micro;L||0.5&micro;L||3&micro;L||0.3&micro;L||23.7&micro;L||30&micro;L
 +
|-
 +
|NC||0.3&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.6&micro;L||10&micro;L
 +
|}
 +
</div>
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
! ||8/28 Plux||SpeI||PstI||BufferD||BSA||MilliQ||total
 +
|-
 +
|2 cuts||6&micro;L||0.5&micro;L||0.5&micro;L||3&micro;L||0.3&micro;L||19.7&micro;L||30&micro;L
 +
|-
 +
|NC||1&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 RBS-tetR-DT-(1)||XbaI||PstI||BufferD||BSA||MilliQ||total
 +
|-
 +
|2 cuts||5&micro;L||0.5&micro;L||0.5&micro;L||3&micro;L||0.3&micro;L||20.7&micro;L||30&micro;L
 +
|-
 +
|NC||0.5&micro;L||0&micro;L||0&micro;L||1&micro;L||0.1&micro;L||8.4&micro;L||10&micro;L
 +
|}
 +
*incubated 37&deg;C 1hour
 +
</div>
 +
===Electrophoresis===
===Electrophoresis===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme1||Enzyme2
 +
|-
 +
|1||8/22 RBS-lysis1-(1)||EcoRI||SpeI
 +
|-
 +
|2||8/22 RBS-lysis1-(1)||--||--
 +
|-
 +
|3||8/17 RBS-GFP-DT-(2)||XbaI||PstI
 +
|-
 +
|4||8/17 RBS-GFP-DT-(2)||--||--
 +
|-
 +
|5||8/20 Pcon-RBS-GFP-DT-(1)||EcoRI||SpeI
 +
|-
 +
|6||8/20 Pcon-RBS-GFP-DT-(1)||--||--
 +
|-
 +
|7||100bp ladder||--||--
 +
|-
 +
|8||8/20 Pcon-RBS-luxR-DT-(1)||EcoRI||XbaI
 +
|-
 +
|9||8/20 Pcon-RBS-luxR-DT-(1)||--||--
 +
|-
 +
|10||8/28 Plux||SpeI||PstI
 +
|-
 +
|11||8/28 Plux||--||--
 +
|-
 +
|12||8/20 RBS-tetR-DT||XbaI||PstI
 +
|-
 +
|13||8/20 RBS-tetR-DT||--||--
 +
|}
 +
[[File:Igku Aug28 Electrophoresis(ColoP)(N4)pic.jpg]]<br>
 +
</div>
 +
===Gel Extraction===
===Gel Extraction===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1||100bp ladder||--
 +
|-
 +
|2||rowspan="2"|8/20 RBS-lysis1-(1)||rowspan="2"|EcoRI & SpeI
 +
|-
 +
|3
 +
|-
 +
|4||--||--
 +
|-
 +
|5||rowspan="2"|8/28 RBS-GFP-DT-(2)||rowspan="2"|XbaI & PstI
 +
|-
 +
|6
 +
|}
 +
[[File:Igku Aug28 Gel Extraction(N⑤-1,2)before.jpg]]<br>
 +
[[File:Igku Aug28 Gel Extraction4-2.jpg]]<br>
 +
{| class="wikitable"
 +
!Lane||DNA||Enzyme
 +
|-
 +
|1||100bp ladder||--
 +
|-
 +
|2||rowspan="2"|8/28 Plux||rowspan="2"|SpeI & PstI
 +
|-
 +
|3
 +
|-
 +
|4||--||--
 +
|-
 +
|5||rowspan="2"|8/20 RBS-tetR-DT-(1)||rowspan="2"|XbaI & PstI
 +
|-
 +
|6
 +
|}
 +
[[File:Igku Aug28 Gel Extraction(N⑤-3).jpg]]<br>
 +
{| class="wikitable"
 +
!Name||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|Pcon-RBS-GFP-DT (EcoRI&SpeI)||7.1||1.88||0.07
 +
|-
 +
|Pcon-RBS-luxR-DT (EcoRI&XbaI)||15.3||1.93||0.70
 +
|-
 +
|Plux (SpeI&PstI)||4.5||1.79||0.31
 +
|-
 +
|RBS-GFP-DT (XbaI&PstI)||6.0||2.16||0.03
 +
|-
 +
|RBS-tetR-DT (XbaI&PstI)||7.6||1.91||0.38
 +
|}
 +
</div>
 +
===Colony PCR===
===Colony PCR===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!|Sample||base pair
 +
|-
 +
|8/27 pT181 attenuator-DT-1||738
 +
|-
 +
|8/27 pT181 attenuator-DT-2||738
 +
|-
 +
|8/27 pT181 attenuator-DT-3||738
 +
|-
 +
|8/27 TetR-aptamer 12_1R-DT-1||521
 +
|-
 +
|8/27 TetR-aptamer 12_1R-DT-2||521
 +
|-
 +
|8/27 pT181 antisense-DT-1||542
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
 +
|-
 +
|5min||30s||30s||30s||30cycles
 +
|}
 +
[[File:Igku Aug28 c(N⑤-1,2)before.jpg]]
 +
{| class="wikitable"
 +
!|Sample||base pair
 +
|-
 +
|8/27 P&lambda;-luxI-1||--
 +
|-
 +
|8/27 P&lambda;-luxI||--
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
 +
|-
 +
|5min||30s||30s||30min||3cycles
 +
|}
 +
[[File:Igku Aug28 ColonyPCR(N⑦-2).jpg]]
 +
</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===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Sample||medium
 +
|-
 +
|8/16 Master Plate(CP)-6 DT||Plusgrow medium(+CP)
 +
|-
 +
|J23100 Master Plate-1||Plusgrow medium(+Amp)
 +
|-
 +
|Plac Master Plate-1||Plusgrow medium(+CP)
 +
|}
 +
</div>
 +
===Ligation===
===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)||2.9||8/28 RBS-lysis1 (EcoRI & SpeI)||9.5
 +
|-
 +
|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||8/27 DT (EcoRI & XbaI)||2.7||8/24 Spinach (EcoRI & SpeI)||2.4
 +
|}
 +
*Samples were evaporeted used evaporator into about 1 &micro;L.
 +
{| class="wikitable"
 +
!sample||MilliQ||Ligation High||total
 +
|-
 +
|1||3||4||9
 +
|}
 +
*incubate overnight at 4 &deg;C
 +
</div>
 +
===Liquid Culture===
===Liquid Culture===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Sample||medium
 +
|-
 +
|8/26 P&lambda;-RBS-luxI-DT-1||Plusgrow medium (+Amp)
 +
|-
 +
|8/26 P&lambda;-RBS-luxI-DT-2||Plusgrow medium (+Amp)
 +
|}
 +
* incubate 37&deg;C 10hour
 +
</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
Retrieved from "http://2013.igem.org/Template:Kyoto/Notebook/Aug_28"