Template:Kyoto/Notebook/Sep 5

From 2013.igem.org

(Difference between revisions)
(→Electrophoresis)
(→Gel Extraction)
Line 412: Line 412:
!Name||concentration[µg/mL]||260/280||260/230
!Name||concentration[µg/mL]||260/280||260/230
|-
|-
-
|RBS-lysis1-DT (XbaI & PstI)||5.3||1.98||0.06
+
|Pconst||5.3||1.98||0.06
-
|-
+
-
|RBS-lysis3-DT (XbaI & PstI)||5.3||1.98||0.06
+
|}
|}
{| class="wikitable"
{| class="wikitable"
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|1||100bp ladder||
|1||100bp ladder||
|-
|-
-
|3||Pcon-pT181 attenuator||EcoRI & SpeI
+
|2||Plac||SpeI & PstI
|-
|-
-
|4||Pcon-pT181 attenuator||EcoRI & SpeI
+
|3||Plac||SpeI & PstI
|-
|-
-
|6||Spinach-DT||XbaI & PstI
+
|5||Plux||SpeI & PstI
|-
|-
-
|7||Spinach-DT||XbaI & PstI
+
|6||Plux||SpeI & PstI
-
|-
+
-
|9||RBS-lacZα-DT||XbaI & PstI
+
-
|-
+
-
|10||RBS-lacZα-DT||XbaI & PstI
+
|}
|}
[[File:igku_xxbeforexx.xxx]]<br>
[[File:igku_xxbeforexx.xxx]]<br>
Line 438: Line 432:
!Name||concentration[&micro;g/mL]||260/280||260/230
!Name||concentration[&micro;g/mL]||260/280||260/230
|-
|-
-
|Pcon-pT181 attenuator (EcoRI & SpeI)||5.2||1.82||0.36
+
|Plac (SpeI & PstI)||5.2||1.82||0.36
|-
|-
-
|Spinach-DT (XbaI & PstI)||8.0||1.87||0.25
+
|Plux(SpeI & PstI)||8.0||1.87||0.25
-
|-
+
-
|RBS-lacZ&alpha;-DT (XbaI & PstI)||10.3||1.71||0.54
+
|}
|}
{| class="wikitable"
{| class="wikitable"
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|1||1kbp ladder||
|1||1kbp ladder||
|-
|-
-
|2||Ptet-pT181 antisense||EcoRI & PstI
+
|2||Pconst-RBS-luxR-DT||EcoRI & XbaI
|-
|-
-
|3||Ptet-pT181 antisense||EcoRI & PstI
+
|3||Pconst-RBS-luxR-DT||EcoRI & XbaI
|-
|-
-
|5||pSB4K5||EcoRI & SpeI
+
|5||Plux-RBS-GFP-DT||EcoRI & SpeI
|-
|-
-
|6||pSB4K5||EcoRI & SpeI
+
|6||Plux-RBS-GFP-DT||EcoRI & SpeI
|}
|}
[[File:igku_xxbeforexx.xxx]]<br>
[[File:igku_xxbeforexx.xxx]]<br>
Line 462: Line 454:
!Name||concentration[&micro;g/mL]||260/280||260/230
!Name||concentration[&micro;g/mL]||260/280||260/230
|-
|-
-
|Ptet-pT181 antisense(EcoRI & PstI)||30.6||1.84||1.16
+
|Pconst-RBS-luxR-DT(EcoRI & XbaI)||30.6||1.84||1.16
-
|-
+
-
|pSB4K5 (EcoRI & SpeI)||21.8||1.87||0.98
+
-
|}
+
-
{| class="wikitable"
+
-
!Lane||DNA||Enzyme
+
-
|-
+
-
|1||1kbp ladder||
+
-
|-
+
-
|2||Pcon-pT181 attenuator||SpeI & PstI
+
-
|-
+
-
|3||Pcon-pT181 attenuator||SpeI & PstI
+
-
|-
+
-
|5||Plac||SpeI & PstI
+
-
|-
+
-
|6||Plac||SpeI & PstI
+
-
|-
+
-
|8||pSB1C3||EcoRI & SpeI
+
-
|-
+
-
|9||pSB1C3||EcoRI & SpeI
+
-
|-
+
-
|11||pSB1C3||XbaI & PstI
+
-
|-
+
-
|12||pSB1C3||XbaI & PstI
+
-
|}
+
-
[[File:igku_xxbeforexx.xxx]]<br>
+
-
[[File:igku_xxafterxx.xxx]]<br>
+
-
{| class="wikitable"
+
-
!Name||concentration[&micro;g/mL]||260/280||260/230
+
-
|-
+
-
|Pcon-pT181 attenuator (SpeI & PstI)||14.5||1.91||1.09
+
-
|-
+
-
|Plac (SpeI & PstI)||11.6||1.74||1.19
+
-
|-
+
-
|pSB1C3 (EcoRI & SpeI)||5.7||1.74||0.34
+
|-
|-
-
|pSB1C3 (XbaI & PstI)||5.6||2.00||0.37
+
|Plux-RBS-GFP-DT (EcoRI & SpeI)||21.8||1.87||0.98
|}
|}

Revision as of 19:12, 24 September 2013

Contents

Sep 5

Colony PCR

No name

Samplebase pair
8/9 J23100-31142
8/9 J23100-41142
8/9 J23100-51142
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5m30s30s30s30cycles

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2J23100-3----
3J23100-4----
4J23100-5----
5100bp ladder----

File:Igku xx.xxx

Miniprep

Nakamoto

DNAconcentration[µg/mL]260/280260/230
8/3 J23100-3349.11.691.851
8/10 Plac-2331.31.841.42

Ligation

Kojima

stateVectorInserterLigation High ver.2
experimentPconst13.2spinach-DT2.13.5
experimentPlac15.3spinach-DT2.13.5
experimentPconst13.2pT181 antisense (XbaI & PstI)6.63.5
experimentPlac15.3pT181 antisense (XbaI & PstI)6.63.5
experimentPtet30.1pT181 antisense (XbaI & PstI)6.63.5
experimentPconst13.2pT181 attenuator11.73.5
experimentPlac15.3pT181 attenuator11.73.5
experimentPtet30.1RBS-lacZα-DT3.03.5

incubate 16 °C 1 hour

Gel Extraction

No name

LaneDNAEnzyme
11kbp ladder
2RBS-luxI-DT
3RBS-luxI-DT
41kbp ladder
5Pbad/araC
6Pbad/araC

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Nameconcentration[µg/mL]260/280260/230
RBS-luxI-DT23.20.837.89
Pbad/araC51.81.200.11

Colony PCR

No name

Samplebase pair
9/4 apt12_1R(pSB1C3)-1384
9/4 apt12_1R(pSB1C3)-2384
9/4 pT181 antisense(pSB1C3)-1415
9/4 pT181 antisense(pSB1C3)-2415
9/4 pT181 antisense(pSB1C3)-3415
9/4 pT181 antisense(pSB1C3)-4415
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30s30cycles
Samplebase pair
9/4 apt12_1R-DT-1521
9/4 apt12_1R-DT-2521
9/4 apt12_1R-DT-3521
9/4 apt12_1R-DT-4521
9/4 pT181 attenuator(pSB1C3)601
9/4 pT181 attenuator(pSB1C3)601
9/4 RBS-lysis1-DT613
9/4 RBS-lysis1-DT613
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s36s30cycles
Samplebase pair
9/4 RBS-lysis2-DT-1985
9/4 RBS-lysis2-DT-2985
9/4 RBS-lysis2-DT-3985
9/4 RBS-lysis2-DT-4985
9/4 RBS-lysis3-DT-11210
9/4 RBS-lysis3-DT-21210
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s72s30cycles

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2apt12_1R (pSB1C3)-1----
3apt12_1R (pSB1C3)-2----
4pT181 antisense(pSB1C3)-1----
5pT181 antisense(pSB1C3)-2----
6pT181 antisense(pSB1C3)-3----
7pT181 antisense(pSB1C3)-4----
8100bp ladder----

File:Igku xx.xxx

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2apt12_1R-DT-1----
3apt12_1R-DT-2----
4apt12_1R-DT-3----
5apt12_1R-DT-4----
6pT181 attenuator(pSB1C3)-1----
7pT181 attenuator(pSB1C3)-2----
8RBS-lysis1-DT-1----
9RBS-lysis1-DT-2----
10100bp ladder----

File:Igku xx.xxx
</div>

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2RBS-lysis2-DT-1----
3RBS-lysis2-DT-2----
4RBS-lysis2-DT-3----
5RBS-lysis2-DT-4----
6RBS-lysis3-DT-1----
7RBS-lysis3-DT-2----
8100bp ladder----

File:Igku xx.xxx
</div>

Liquid Culture

Nakamoto

Samplemedium
8/9 J23100-5Plusgrow medium(+Amp)

37°C

Transformation

No name

NameSample(µL)Competent Cells(µL)Total(µL)Plate
9/5 Pconst+spinach-DT22022--
9/5 Plac+spinach-DT22022--
9/5 Pconst+pT181 antisense22022--
9/5 Plac+pT181 antisense22022--
9/5 Ptet+pT181 antisense22022--
9/5 Plux+pT181 attenuater22022--
9/5 Ptet+pT181 attenuater22022--
9/5 Ptet+pT181 attenuater22022--
9/5 Pconst+RBS-tetR+DT22022--
9/16(2012) T7-His-FT22022--
9/16(2012) pBr32222022--

Restriction Enzyme Digestion

No name

9/5 Pbad/araCESXPBSABufferMilliQtotal
2 cuts4µL 0µL 1µL 0µL 1µL 3µL 3µL 18µL 30µL
9/5 RBS-luxI-DTESXPBSABufferMilliQtotal
2 cuts4µL 0µL 0µL 1µL 1µL 3µL 3µL18µL 30µL
8/28 PluxESXPBSABufferMilliQtotal
2 cuts 12µL 0µL 1µL 0µL 1µL 3µL 3µL10µL30µL
NC 0.6µL0µL0µL 0µL 0µL1µL1µL7.4µL10µL
9/5 Pconst(J23100)ESXPBSABufferMilliQtotal
2 cuts5.7µL0µL1µL0µL1µL3µL3µL16.3µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
9/5 PlacESXPBSABufferMilliQtotal
2 cuts6.0µL0µL1µL0µL1µL3µL3µL16µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/31 Plux-RBS-GFP-DT-1ESXPBSABufferMilliQtotal
2 cuts9.4µL1µL1µL0µL0µL3µL3µL12.6µL30µL
NC 0.5µL0µL0µL 0µL 0µL1µL1µL7.5µL10µL
8/20 Pconst-RBS-luxR-DTESXPBSABufferMilliQtotal
2 cuts4.5µL1µL0µL1µL0µL3µL3µL13.5µL30µL
NC 0.2µL0µL0µL 0µL 0µL1µL1µL7.8µL10µL

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
11kbp ladder----
2PconstSpeIPstI
3Pconst----
4PlacSpeIPstI
5Plac----
6PluxSpeIPstI
7Plux----
8Pconst-RBS-luxR-DTEcoRIXbaI
9Pconst-RBS-luxR-DT----
10Plux-RBS-GFP-DTEcoRISpeI
11Plux-RBS-GFP-DT----
121kbp ladder----

File:Igku xx.xxx

5*M9(EDTA)

Hirano

volume10ml
Na2HPO4 60mg
KH2PO430mg
NaCl5mg
NH4Cl10mg
Fe(3)-EDTA1263.27g　

121°C 20minute autoclave

Gel Extraction

No name

LaneDNAEnzyme
11kbp ladder
2PconstSpeI & PstI
3PconstSpeI & PstI

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Nameconcentration[µg/mL]260/280260/230
Pconst5.31.980.06
LaneDNAEnzyme
1100bp ladder
2PlacSpeI & PstI
3PlacSpeI & PstI
5PluxSpeI & PstI
6PluxSpeI & PstI

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Nameconcentration[µg/mL]260/280260/230
Plac (SpeI & PstI)5.21.820.36
Plux(SpeI & PstI)8.01.870.25
LaneDNAEnzyme
11kbp ladder
2Pconst-RBS-luxR-DTEcoRI & XbaI
3Pconst-RBS-luxR-DTEcoRI & XbaI
5Plux-RBS-GFP-DTEcoRI & SpeI
6Plux-RBS-GFP-DTEcoRI & SpeI

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Nameconcentration[µg/mL]260/280260/230
Pconst-RBS-luxR-DT(EcoRI & XbaI)30.61.841.16
Plux-RBS-GFP-DT (EcoRI & SpeI)21.81.870.98

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
Pcon-pT181 anti243.01.901.81
Pcon-Spinach-DT253.71.571.51

Colony PCR

No name

Samplebase pair
9/8 Plac-spinach-DT-(1)659
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5m30s30s40s30cycles
Samplebase pair
9/8 Plac-spinach-DT-(2)659
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5m30s30s40s30cycles
Samplebase pair
9/8 Pcon-pt181atte-DT-(1)584
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5m30s30s40s30cycles

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder----
28/19 lysis1XbaIPstI
38/19 lysis1XbaI--
48/19 lysis1--PstI
58/19 lysis1----
68/19 lysis1(Gel Extraction Product)XbaIPstI

Liquid Culture

Hirano

Samplemedium
JM109-1(Master plate)LB medium
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