Template:Kyoto/Notebook/Sep 5

From 2013.igem.org

(Difference between revisions)
(→Colony PCR not yet)
(→Colony PCR)
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===Colony PCR===
===Colony PCR===
<div class="experiment">
<div class="experiment">
-
<span class="author">Hirano</span>
+
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
!Sample||base pair
!Sample||base pair
|-
|-
-
|8/9 BBa_J23100 3||--
+
|9/4 apt12_1R(pSB1C3)-1||384
|-
|-
-
|8/9 BBa_J23100 4||--
+
|9/4 apt12_1R(pSB1C3)-2||384
|-
|-
-
|8/9 BBa_J23100 5||--
+
|9/4 pT181 antisense(pSB1C3)-1||415
|-
|-
 +
|9/4 pT181 antisense(pSB1C3)-2||415
 +
|-
 +
|9/4 pT181 antisense(pSB1C3)-3||415
 +
|-
 +
|9/4 pT181 antisense(pSB1C3)-4||415
|}
|}
{| class="wikitable"
{| class="wikitable"
Line 128: Line 133:
|-
|-
|5min||30s||30s||30s||30cycles
|5min||30s||30s||30s||30cycles
 +
|}
 +
</div>
 +
{| class="wikitable"
 +
!Sample||base pair
 +
|-
 +
|9/4 apt12_1R-DT-1||521
 +
|-
 +
|9/4 apt12_1R-DT-2||521
 +
|-
 +
|9/4 apt12_1R-DT-3||521
 +
|-
 +
|9/4 apt12_1R-DT-4||521
 +
|-
 +
|9/4 pT181 attenuator(pSB1C3)||601
 +
|-
 +
|9/4 pT181 attenuator(pSB1C3)||601
 +
|-
 +
|9/4 RBS-lysis1-DT||613
 +
|-
 +
|9/4 RBS-lysis1-DT||613
 +
|-
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
 +
|-
 +
|5min||30s||30s||36s||30cycles
 +
|}
 +
</div>
 +
{| class="wikitable"
 +
!Sample||base pair
 +
|-
 +
|9/4 RBS-lysis2-DT-1||985
 +
|-
 +
|9/4 RBS-lysis2-DT-2||985
 +
|-
 +
|9/4 RBS-lysis2-DT-3||985
 +
|-
 +
|9/4 RBS-lysis2-DT-4||985
 +
|-
 +
|9/4 RBS-lysis3-DT-1||1210
 +
|-
 +
|9/4 RBS-lysis3-DT-2||1210
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
 +
|-
 +
|5min||30s||30s||72s||30cycles
|}
|}
</div>
</div>

Revision as of 17:47, 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
Pcon-pT181 attenuator (SpeI & PstI)14.51.911.09
Plac (SpeI & PstI)11.61.741.19
pSB1C3 (EcoRI & SpeI)5.71.740.34
pSB1C3 (XbaI & PstI)5.62.000.37

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

Liquid Culture

Nakamoto

Samplemedium
9/11Fusion6 antisense-1Plusgrow medium(+CP)
Plac(BBa-R0011)-3Plusgrow medium(+Amp)
Plac(BBa-R0011)-4Plusgrow medium(+Amp)
Fusion3m2 attenuator -1Plusgrow medium(+CP)　
Pλ-luxI-2Plusgrow medium(+Amp)　
9/11 aptamer12-1M-2Plusgrow medium(+CP)

Transformation

じっけんしゃ

NameSample(µL)Competent Cells(µL)Total(µL)Plate
9/8 Pcont+aptamer 12-1R-DT11011Amp
9/8 Pcon+RBS-lacZα-DT11011Amp
9/8 Pcon-pT181 attenuater+aptamer 12-1R-DT11011Amp
9/8 Plac+RBS-lysis1-DT22022CP
9/8 Plac+RBS-lysis2-DT22022CP
9/8 Plac+aptamer 12-1R-DT22022CP
9/8 Plac+spinach22022CP
9/8 Plac+pT181 attenuater22022CP
9/8 Plux+RBS-lysis1-DT11011CP
9/8 Plux+RBS-lysis2-DT11011CP
9/8 Ptet+RBS-lacZα-DT11011CP
9/8 Ptet-pT181 anti+spinach-DT11011CP
9/8 Pcon-pT181 attenuater+DT11011CP

Restriction Enzyme Digestion

じっけんしゃ

9/8 Pcon-pT181 antiESXPBSABufferMilliQtotal
2 cuts8.2µL 0µL 1µL 0µL 1µL 3µL 3µL 13.8µL 30µL
NC 0.4µL 0µL 0µL 0µL 0µL 1µL 1µL 7.6µL 10µL
9/8 Pcon-spin-DTESXPBSABufferMilliQtotal
2 cuts7.9µL 1µL 1µL 0µL 0µL 3µL 3µL14.1µL 30µL
NC 0.4µL 0µL 0µL 0µL 0µL 1µL 1µL7.6µL 10µL
8/21 F1 atteESXPBSABufferMilliQtotal
2 cuts 6.5µL 0µL 0µL 1µL 1µL 3µL 3µL15.5µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/21 F3m2 atteESXPBSABufferMilliQtotal
2 cuts6.0µL0µL0µL1µL1µL3µL3µL16µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/21 F1 antiESXPBSABufferMilliQtotal
2 cuts6.7µL0µL0µL1µL1µL3µL3µL15.3µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/21 F6 antiESXPBSABufferMilliQtotal
2 cuts6.0µL0µL0µL1µL1µL3µL3µL16µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/21 tetR aptamer 12_1MESXPBSABufferMilliQtotal
2 cuts5.3µL1µL1µL0µL0µL3µL3µL16.7µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/21 tetR aptamer 12_PESXPBSABufferMilliQtotal
2 cuts13.3µL1µL1µL0µL0µL3µL3µL8.7µL30µL
NC0.7µL0µL0µL0µL0µL1µL1µL7.3µL10µL
9/4 spin-DTESXPBSABufferMilliQtotal
2 cuts5.4µL0µL0µL1µL1µL3µL3µL16.6µL30µL
NC0.3µL0µL0µL0µL0µL1µL1µL7.7µL10µL
9/7 RBS-lysis1-DTESXPBSABufferMilliQtotal
2 cuts10µL0µL0µL1µL1µL3µL3µL12µL30µL
NC0.5µL0µL0µL0µL0µL1µL1µL7.5µL10µL
9/6 RBS-lysis2-DTESXPBSABufferMilliQtotal
2 cuts13µL0µL0µL1µL1µL3µL3µL9µL30µL
NC0.7µL0µL0µL0µL0µL1µL1µL7.3µL10µL
9/7 RBS-lysis3-DTESXPBSABufferMilliQtotal
2 cuts10µL0µL0µL1µL1µL3µL3µL12µL30µL
NC0.5µL0µL0µL0µL0µL1µL1µL7.5µL10µL
8/20 J23100 RBS-lacZα-DT-(1)ESXPBSABufferMilliQtotal
2 cuts7µL0.5µL0.5µL0µL0µL3µL3µL16µL30µL
NC0.5µL0µL0µL0µL0µL1µL1µL7.5µL10µL
8/20 RBS-lacZα-DTESXPBSABufferMilliQtotal
2 cuts10µL0.5µL0.5µL0µL0µL3µL3µL13µL30µL
NC0.5µL0µL0µL0µL0µL1µL1µL7.5µL10µL

5*M9(EDTA)

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder
2RBS-lysis1-DTXbaI & PstI
3RBS-lysis1-DTXbaI & PstI
5RBS-lysis3-DTXbaI & PstI
6RBS-lysis3-DTXbaI & PstI

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

Nameconcentration[µg/mL]260/280260/230
RBS-lysis1-DT (XbaI & PstI)5.31.980.06
RBS-lysis3-DT (XbaI & PstI)5.31.980.06
LaneDNAEnzyme
1100bp ladder
3Pcon-pT181 attenuatorEcoRI & SpeI
4Pcon-pT181 attenuatorEcoRI & SpeI
6Spinach-DTXbaI & PstI
7Spinach-DTXbaI & PstI
9RBS-lacZα-DTXbaI & PstI
10RBS-lacZα-DTXbaI & PstI

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

Nameconcentration[µg/mL]260/280260/230
Pcon-pT181 attenuator (EcoRI & SpeI)5.21.820.36
Spinach-DT (XbaI & PstI)8.01.870.25
RBS-lacZα-DT (XbaI & PstI)10.31.710.54
LaneDNAEnzyme
11kbp ladder
2Ptet-pT181 antisenseEcoRI & PstI
3Ptet-pT181 antisenseEcoRI & PstI
5pSB4K5EcoRI & SpeI
6pSB4K5EcoRI & SpeI

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

Nameconcentration[µg/mL]260/280260/230
Ptet-pT181 antisense(EcoRI & PstI)30.61.841.16
pSB4K5 (EcoRI & SpeI)21.81.870.98
LaneDNAEnzyme
11kbp ladder
2Pcon-pT181 attenuatorSpeI & PstI
3Pcon-pT181 attenuatorSpeI & PstI
5PlacSpeI & PstI
6PlacSpeI & PstI
8pSB1C3EcoRI & SpeI
9pSB1C3EcoRI & SpeI
11pSB1C3XbaI & PstI
12pSB1C3XbaI & PstI

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

Nameconcentration[µg/mL]260/280260/230
Pcon-pT181 attenuator (SpeI & PstI)14.51.911.09
Plac (SpeI & PstI)11.61.741.19
pSB1C3 (EcoRI & SpeI)5.71.740.34
pSB1C3 (XbaI & PstI)5.62.000.37

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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