Template:Kyoto/Notebook/Sep 5

From 2013.igem.org

(Difference between revisions)
(→Colony PCR)
(→Restriction Enzyme Digestion)
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<span class="author">No name</span>
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
-
! ||9/5 Pbad/araC||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||9/5 Pbad/araC||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts||4&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L|| 18&micro;L|| 30&micro;L
|2 cuts||4&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L|| 18&micro;L|| 30&micro;L
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||9/5 RBS-luxI-DT||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||9/5 RBS-luxI-DT||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts||4&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L||18&micro;L|| 30&micro;L
|2 cuts||4&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L||18&micro;L|| 30&micro;L
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||8/28 Plux||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||8/28 Plux||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts|| 12&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L||10&micro;L||30&micro;L
|2 cuts|| 12&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L||10&micro;L||30&micro;L
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|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||9/5 Pconst(J23100)||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||9/5 Pconst(J23100)||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts||5.7&micro;L||0&micro;L||1&micro;L||0&micro;L||1&micro;L||3&micro;L||3&micro;L||16.3&micro;L||30&micro;L
|2 cuts||5.7&micro;L||0&micro;L||1&micro;L||0&micro;L||1&micro;L||3&micro;L||3&micro;L||16.3&micro;L||30&micro;L
Line 250: Line 250:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||9/5 Plac||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||9/5 Plac||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts||6.0&micro;L||0&micro;L||1&micro;L||0&micro;L||1&micro;L||3&micro;L||3&micro;L||16&micro;L||30&micro;L
|2 cuts||6.0&micro;L||0&micro;L||1&micro;L||0&micro;L||1&micro;L||3&micro;L||3&micro;L||16&micro;L||30&micro;L
Line 257: Line 257:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||8/31 Plux-RBS-GFP-DT-1||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||8/31 Plux-RBS-GFP-DT-1||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts||9.4&micro;L||1&micro;L||1&micro;L||0&micro;L||0&micro;L||3&micro;L||3&micro;L||12.6&micro;L||30&micro;L
|2 cuts||9.4&micro;L||1&micro;L||1&micro;L||0&micro;L||0&micro;L||3&micro;L||3&micro;L||12.6&micro;L||30&micro;L
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|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||8/20 Pconst-RBS-luxR-DT||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||8/20 Pconst-RBS-luxR-DT||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts||4.5&micro;L||1&micro;L||0&micro;L||1&micro;L||0&micro;L||3&micro;L||3&micro;L||13.5&micro;L||30&micro;L
|2 cuts||4.5&micro;L||1&micro;L||0&micro;L||1&micro;L||0&micro;L||3&micro;L||3&micro;L||13.5&micro;L||30&micro;L

Revision as of 01:30, 27 September 2013

Contents

Sep 5

Electrophoresis

No name

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

Igku Sep5 electrophoresis N1.jpg

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

Electrophoresis

Tatsui

LaneSample
1100bp ladder
2RBS-luxI-DT
3Pbad/araC
4NC
5100bp ladder

Igku Sep5 electrophorsis N2 1.jpg
Igku Sep5 electrophorsis N2 2.jpg

Gel Extraction

No name

LaneDNAEnzyme
11kbp ladder
2RBS-luxI-DT
3RBS-luxI-DT
4
5Pbad/araC
6Pbad/araC

Igku Sep5 Gel Extraction(N3) 1.jpg
Igku Sep5 Gel Extraction(N3) 2.jpg

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 aptamer 12_1R(pSB1C3)-1384
9/4 aptamer 12_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 aptamer 12_1R-DT-1521
9/4 aptamer 12_1R-DT-2521
9/4 aptamer 12_1R-DT-3521
9/4 aptamer 12_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

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 attenuator22022--
9/5 Ptet+pT181 attenuator22022--
9/5 Ptet+pT181 attenuator22022--
9/5 Pconst+RBS-tetR+DT22022--
9/16(2012) T7-His-FT22022--
9/16(2012) pBr32222022--

Restriction Enzyme Digestion

No name

9/5 Pbad/araCEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts4µL 0µL 1µL 0µL 1µL 3µL 3µL 18µL 30µL
9/5 RBS-luxI-DTEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts4µL 0µL 0µL 1µL 1µL 3µL 3µL18µL 30µL
8/28 PluxEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts 12µL 0µL 1µL 0µL 1µL 3µL 3µL10µL30µL
NC 0.6µL0µL0µL 0µL 0µL1µL1µL7.4µL10µL
9/5 Pconst(J23100)EcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts5.7µL0µL1µL0µL1µL3µL3µL16.3µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
9/5 PlacEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts6.0µL0µL1µL0µL1µL3µL3µL16µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/31 Plux-RBS-GFP-DT-1EcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts9.4µL1µL1µL0µL0µL3µL3µL12.6µL30µL
NC 0.5µL0µL0µL 0µL 0µL1µL1µL7.5µL10µL
8/20 Pconst-RBS-luxR-DTEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts4.5µL1µL0µL1µL0µL3µL3µL13.5µL30µL
NC 0.2µL0µL0µL 0µL 0µL1µL1µL7.8µL10µL
DNAconcentration[µg/mL]260/280260/230
9/5 RBS-luxI-DT7.61.56-1.42
9/5 Pbad/araC7.51.543.84

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2aptamer 12_1R (pSB1C3)-1----
3aptamer 12_1R (pSB1C3)-2----
4pT181 antisense(pSB1C3)-1----
5pT181 antisense(pSB1C3)-2----
6pT181 antisense(pSB1C3)-3----
7pT181 antisense(pSB1C3)-4----
8100bp ladder----

Igku Sep5 Electrophoresis(N4).jpg

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2aptamer 12_1R-DT-1----
3aptamer 12_1R-DT-2----
4aptamer 12_1R-DT-3----
5aptamer 12_1R-DT-4----
6pT181 attenuator(pSB1C3)-1----
7pT181 attenuator(pSB1C3)-2----
8RBS-lysis1-DT-1----
9RBS-lysis1-DT-2----
10100bp ladder----

Igku Sep5 Electrophoresis(N6) 2.jpg

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----

Igku Sep5 Electrophoresis(N5).jpg

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

Igku Sep5 Electrophoresis(N6) 3.jpg

5x M9 Medium (+EDTA)

Hirano

volume10ml
Na2HPO4 60mg
KH2PO430mg
NaCl5mg
NH4Cl10mg
Fe(III)-EDTA1263.27mg　
MilliQup to 10 mL
  • autoclave at 121 °C for 20 min

Gel Extraction

No name

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

Igku Sep5 gel extraction N7 1.jpg
Igku Sep5 gel extraction N7 2.jpg

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

Igku Sep5 gel extraction N7 3.jpg
Igku Sep5 gel extraction N7 4.jpg

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

Igku Sep5 gel extraction N7 5.jpg
Igku Sep5 gel extraction N7 6.jpg

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

DNA Purification

No name

DNAconcentration[µg/mL]260/280260/230
9/5 RBS-luxI-DT7.61.56-1.42
9/5 Pbad/araC7.51.543.84

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
8/9 J23100-5243.01.901.81

Colony PCR

No name

Samplebase pair
9/4 RBS-lysis3-DT-31210
9/4 RBS-lysis3-DT-41210
9/4 RBS-lysis3-DT-51210
9/4 RBS-lysis1-DT-3613
9/4 Ptrc KaiC-1--
9/4 pSB4K51370
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min 25s30cycles

FileIgku Sep6 ColonyPCRN1.jpg

Liquid Culture

Hirano

Samplemedium
9/4 aptamer 12_1R-DT-1Plusgrow medium (+CP)
9/4 pT181 attenuator(pSB1C3)-1Plusgrow medium (+CP)
9/4 RBS-lysis2-DT-1Plusgrow medium (+CP)
9/4 Ptrc-KaiC -1Plusgrow medium (+Amp)
9/4 pSB4K5 -1Plusgrow medium (+Kan)
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