Template:Kyoto/Notebook/Sep 12

From 2013.igem.org

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(→Electrophoresis)
(→Colony PCR)
 
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Line 6: Line 6:
!DNA||concentration[µg/mL]||260/280||260/230  
!DNA||concentration[µg/mL]||260/280||260/230  
|-
|-
-
|Pcon-pT181atte-DT||293.6　||1.88　||1.84
+
|Pcon-pT181attenuator-DT||293.6　||1.88　||1.84
|-
|-
|Pcon-apt12-1R-DT||303.8　||1.75　||1.64
|Pcon-apt12-1R-DT||303.8　||1.75　||1.64
|-
|-
-
|Fusion1atte(pSB1c3)||172.0　||1.96　||2.31
+
|Fusion1attenuator(pSB1c3)||172.0　||1.96　||2.31
|-
|-
|RBS-lisis1-DT||235.1||1.59||1.60
|RBS-lisis1-DT||235.1||1.59||1.60
Line 43: Line 43:
|9/10 Pcon-apt12-1R-DT1||Plusgrow medium(+Amp)
|9/10 Pcon-apt12-1R-DT1||Plusgrow medium(+Amp)
|-
|-
-
|9/10 DT-Pcon-pT181atte1||Plusgrow medium(+CP)
+
|9/10 DT-Pcon-pT181attenuator1||Plusgrow medium(+CP)
|-
|-
-
|9/10 Fusion1-atte(pSB1c3)||Plusgrow medium(+CP)　
+
|9/10 Fusion1-attenuator(pSB1c3)||Plusgrow medium(+CP)　
|}
|}
</div>
</div>
Line 53: Line 53:
<span class="author">Tatsui</span></div>
<span class="author">Tatsui</span></div>
-
1
 
{| class="wikitable"
{| class="wikitable"
!Lane||Sample
!Lane||Sample
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|4||RBS-lacz-DT(EcoRI&SpeI)
|4||RBS-lacz-DT(EcoRI&SpeI)
|-
|-
-
|5|| EcoR1&Spe1 NC( EcoRI&SpeI)
+
|5|| EcoRI&SpeI NC( EcoRI&SpeI)
|-
|-
|6||Pcon-RBS-lacz-DT( EcoRI&SpeI)
|6||Pcon-RBS-lacz-DT( EcoRI&SpeI)
Line 85: Line 84:
|1||1kb ladder
|1||1kb ladder
|-
|-
-
|2||Ptet-pT181anti(SpeI&PstI)  
+
|2||Ptet-pT181antisense(SpeI&PstI)  
|-
|-
-
|3|| Ptet-pT181anti NC(SpeI&PstI)
+
|3|| Ptet-pT181antisense NC(SpeI&PstI)
|-
|-
|4||RBS-lysis2-DT(XbaI&PstI)
|4||RBS-lysis2-DT(XbaI&PstI)
|-
|-
-
|5|| EcoR1&Spe1 NC( EcoRI&SpeI)
+
|5|| EcoRI&SpeI NC( EcoRI&SpeI)
|-
|-
|6||--
|6||--
Line 249: Line 248:
===Colony PCR===
===Colony PCR===
<div class="experiment">
<div class="experiment">
-
<span class="author">Nakamoto,Tatsui</span>
+
<span class="author">Nakamoto and Tatsui</span>
{| class="wikitable"
{| class="wikitable"
!Sample||base pair
!Sample||base pair
|-
|-
-
|9/11 Pcon-pT181atte-RBS-lacZ&alpha;-DT||965
+
|9/11 Pcon-pT181attenuator-RBS-lacZ&alpha;-DT||965
|-
|-
|9/11 Plux-PBS-lysis1-DT 1||613
|9/11 Plux-PBS-lysis1-DT 1||613
Line 278: Line 277:
===Transformation===
===Transformation===
<div class="experiment">
<div class="experiment">
-
<span class="author">tatsui</span>
+
<span class="author">Tatsui</span>
{| class="wikitable"
{| class="wikitable"
!Name||Sample||Competent Cells||Plate
!Name||Sample||Competent Cells||Plate
Line 334: Line 333:
|-
|-
|8|| 1kb ladder
|8|| 1kb ladder
-
[[File:igku_91212.png]]<br>
 
|}
|}
 +
[[File:igku_91212.png]]<br>
===Electrophoresis===
===Electrophoresis===
Line 370: Line 369:
|}
|}
</div>
</div>
 +
[[File:igku_91213.png]]
===Liquid Culture===
===Liquid Culture===
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!Lane||Sample||Enzyme
!Lane||Sample||Enzyme
|-
|-
-
|1||Fusion1 anti 2 ||--
+
|1||Fusion1 antisense 2||--
|-
|-
-
|2|| Fusion1 anti 3||--  
+
|2||Fusion1 antisense 3||--  
|-
|-
|3||1kb ladder ||--
|3||1kb ladder ||--
Line 436: Line 436:
===Restriction Enzyme Digestion===
===Restriction Enzyme Digestion===
<div class="experiment">
<div class="experiment">
-
<span class="author">tatsui </span>
+
<span class="author">Tatsui </span>
{| class="wikitable"
{| class="wikitable"
! ||9/12 RBS-lysis2-DT||XbaI||PstI||Buffer||BSA||MilliQ||total
! ||9/12 RBS-lysis2-DT||XbaI||PstI||Buffer||BSA||MilliQ||total

Latest revision as of 20:49, 27 September 2013

Contents

Sep 12

Miniprep

Nakamoto

DNAconcentration[µg/mL]260/280260/230
Pcon-pT181attenuator-DT293.6　1.88　1.84
Pcon-apt12-1R-DT303.8　1.75　1.64
Fusion1attenuator(pSB1c3)172.0　1.96　2.31
RBS-lisis1-DT235.11.591.60
RBS-lisis2-DT387.71.912.01
RBS-lisis3-DT387.91.912.09
Plux166.71.981.63

Electrophoresis

Nakamoto

LaneSample
1100bp ladder
29/11 P-RBS-lisis1-DT(Colony PCR prodution)

Igku 9121.png

Liquid Culture

Nakamoto

Samplemedium
9/10 Pcon-apt12-1R-DT1Plusgrow medium(+Amp)
9/10 DT-Pcon-pT181attenuator1Plusgrow medium(+CP)
9/10 Fusion1-attenuator(pSB1c3)Plusgrow medium(+CP)　

Electrophoresis

Tatsui
LaneSample
11kb ladder
2pSB4K5(EcoRI&SpeI)
3 pSB4K5 NC(EcoRI&SpeI)
4RBS-lacz-DT(EcoRI&SpeI)
5 EcoRI&SpeI NC( EcoRI&SpeI)
6Pcon-RBS-lacz-DT( EcoRI&SpeI)
7 Pcon-RBS-lacz-DT NC( EcoRI&SpeI)
81kb ladder

Igku 9122.png

2

LaneSample
11kb ladder
2Ptet-pT181antisense(SpeI&PstI)
3 Ptet-pT181antisense NC(SpeI&PstI)
4RBS-lysis2-DT(XbaI&PstI)
5 EcoRI&SpeI NC( EcoRI&SpeI)
6--
7--
81kb ladder

Igku 9123.png

Gel Extraction

Tatsui

LaneDNAEnzyme
11kb ladder--
2RBS-lacZα-DTEcoRI&SpeI
3RBS-lacZα-DTEcoRI&SpeI
4RBS-lacZα-DTEcoRI&SpeI
5----
6Pcon-RBS-lacZα-DTEcoRI&SpeI
7Pcon-RBS-lacZα-DTEcoRI&SpeI
8Pcon-RBS-lacZα-DTEcoRI&SpeI
91kb ladder--

Igku 9129.png
Igku 91210.png

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(EcoRI&SpeI)9.81.500.39
Pcon-RBS-lacZα-DT(EcoRI&SpeI)11.21.470.08

Gel Extraction

Tatsui

LaneDNAEnzyme
11kb ladder--
2RBS-lysis2-DTXbaI&PstI
3RBS-lysis2-DTXbaI&PstI
4RBS-lysis2-DTXbaI&PstI
5----
61kb ladder--

Igku 9127.png
Igku 9128.png

Nameconcentration[µg/mL]260/280260/230
RBS-lysis2-DT(XbaI&PstI)5.41.430.77

Electrophoresis

No name


LaneSample
11kb ladder
2Pλ-RBS-luxI-DT(PCR product)
3Pbad/araC-RBS-RFP(PCR product)

Igku 9124.png

Colony PCR

Honda

Samplebase pair
9/11 apt12-1M(pSB1C3) 1513
9/11 apt12-1M(pSB1C3) 2513
9/11 Fusion3m2a09ttenuator(pSB1C3) 1609
9/11 Fusion6 antisense(pSB1C3)1431
9/11 aptamer 12-P(pSB1C3)1515
9/11 Fusion1 antisense(pSB1C3) 1420
9/11 Fusion1 antisense(pSB1C3) 2420
9/11 Plac(BBa-R0011) 1293
9/11 Plac(BBa-R0011) 2293
9/11 Plac(BBa-R0011) 3293
9/11 Plac(BBa-R0011) 4293
9/11 Pcon-luxR-Plux-GFP 12138
9/11 Pcon-RBS-lacZα-DT(pSB4K5) 1712
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s2min12s30cycles

Restriction Enzyme Digestion

Tatsui

9/5 Pcon EcoRISpeIXbaIPstI BufferBSAMilliQtotal
1cuts(PstI)5.70µL0µL0µL1µL3µL3µL17.3µL30µL
NC(PstI)1.90µL0µL0µL0µL1µL1µL6.1µL10µL
8/17 DTEcoRIBufferBSAMilliQtotal
1cut(EcoRI)10.61µL3µL3µL12.4µL30µL
NC(EcoRI)3.50µL1µL1µL4.5µL10µL

Colony PCR

Nakamoto and Tatsui

Samplebase pair
9/11 Pcon-pT181attenuator-RBS-lacZα-DT965
9/11 Plux-PBS-lysis1-DT 1613
9/11 Pcon-pT181attenuator-aptamer12-1R-DT 1859
9/11 Pcon-pT181attenuator-aptamer12-1R-DT 2859
9/11 Plux-RBS-lysis3-DT 11210
9/11 Plux-RBS-lysis3-DT 21210
Pcon-Spinach-DT(pSB4K5) 1605
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min12s30cycles

Transformation

Tatsui

NameSampleCompetent CellsPlate
9/7 pSB4K52µL20µLAmp

Electrophoresis

No name

1

LaneSample
1Fusion6 antisense
2Fusion1 antisense-1
3 Fusion1 antisense-2
4100bp ladder
5Plac(BBa-R0011) 1
6 Plac(BBa-R0011) 2
7 Plac(BBa-R0011) 3
8 Plac(BBa-R0011) 4

Igku 91211.png

2

LaneSample
11kb ladder
2aptamer12-1M 1
3 aptamer12-1M 2
4Fusion3m2 attenuator
5aptamer12-P1
6Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT
7Pcon-RBS-lacZα=DT
8 1kb ladder

Igku 91212.png

Electrophoresis

No name

LaneSampleEnzyme
1Pcon-pT181attenuator-RBS-lacZα-DT 1--
2Pcon-pT181attenuator-RBS-lacZα-DT 2--
3Plux-RBS-lysis1-DT 1 --
4Pcon-pT181attenuator-aptamer12-1R-DT 1 --
5Pcon-pT181attenuator-aptamer12-1R-DT 2 --
6Plux-RBS-lysis3-DT 1--
71kbp ladder --
8 Plux-RBS-lysis3-DT 2--
9Pcon-spinach-DT(pSB4K5) 1--
109/5 PconPstI
119/5 Pcon NC--
128/17 DT EcoRI
138/17 DT --

Igku 91213.png

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)

Colony PCR

Nakamoto

Samplebase pair
9/11 Fusion1 antisense 3394
9/11 Fusion1 antisense 4394
9/11 aptamer12-P 2378
9/11 aptamer12-P 3378
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s36s30cycles


Electrophoresis

Nakamoto

LaneSampleEnzyme
1Fusion1 antisense 2--
2Fusion1 antisense 3--
31kb ladder --
4apt12-P 2 --
5apt12-P 3 --

Igku 91214.png

Restriction Enzyme Digestion

Tatsui

9/12 RBS-lysis2-DTXbaIPstIBufferBSAMilliQtotal
2 cuts5.2 1.0µL1.0µL3.0µL3.0µL16.8µL 30µL
NC0.3µL0µL0µL1.0µL1.0µL7.7µ10µL
9/12 RBS-lysis3-DTXbaI PstIBufferBSAMilliQtotal
2 cuts5.2 1.0µL1.0µL3.0µL3.0µL16.8µL 30µL
NC0.3µL0µL0µL1.0µL1.0µL7.7µ10µL
9/11 RBS-lysis1-DTXbaIPstIBufferBSAMilliQtotal
2 cuts8.51.0µL1.0µL3.0µL3.0µL13.5µL 30µL
NC0.4µL0µL0µL1.0µL1.0µL7.6µ10µL
8/20 J23100-RBS-luxR -DTEcoRI BufferBSAMilliQtotal
1cut5.81.0µL3.0µL3.0µL17.2µL 30µL
NC0.3µL0µL1.0µL1.0µL7.7µ10µL
9/10 pSB1C3EcoRI SpeIBufferBSAMilliQtotal
2cuts8.11.0µL1.0µL3.0µL3.0µL13.9µL 30µL
NC0.4µL0µL0µL1.0µL1.0µL7.6µ10µL
9/12 Plux PstI BufferBSAMilliQtotal
1cut12.01.0µL3.0µL3.0µL11.0micro;L 30µL
NC0.6µL0µL1.0µL1.0µL7.4µ10µL
9/10 pSB1C3XbaI PstIBufferBSAMilliQtotal
2cuts8.11.0µL1.0µL3.0µL3.0µL13.9µL 30µL

Electrophoresis

Tatsui

LaneSampleEnzyme1Enzymel2
19/12 RBS-lysis2-DT XbaIPstI
29/12 RBS-lysis2-DT NC ----
39/11 RBS-lysis1-DT XbaIPstI
49/11 RBS-lysis1-DT NC ----
5 9/11 RBS-lysis3-DT XbaIPstI
6 9/11 RBS-lysis3-DT NC1----
71kb ladder ----
8 100bp ladder----
9J23100-RBS-luxR-DT EcoRI--
10 J23100-RBS-luxR-DT ----
119/10 pSB1C3 EcoRISpeI
12 9/10 pSB1C3 NC-----
139/10 pSB1C3 XbaIPstI
149/12 PluxPstI--
159/12 Plux NC----

Igku 91215.png

Observation through a confocal microscope

We used the liquid culture media with 200μL 9/10Pcon-pT181antisense-spinach-DT& 200μL 9/10 Pcon-spinach-DT& 200μL 9/10 JM109(overnight culture).
We translated them into each 1.5mL tube.

We elminated each supernatant using a 5000xg centrifuge for 1minute,.
Then,we resuspended with 100µL M9(distilled water) and eliminated each supernatant using a 5000xg centrifuge for 1 minute.x2

Adding 100µL M9(in 20µM DFHBI), we resuspended the pret.

15min after incubating in shield light, we eliminated each supernatant using a 5000xg centrifuge for 1 minute.

We looked at the fluorescence of the pret.
We failed to observe it.

Liquid Culture

Hirano

Samplemedium
9/10 Pcon-pT181antisense-spinach-DTPlusgrow medium
9/10 Pcon-spinach-DTPlusgrow medium
9/1 spinach-DT(RNA master plate) Plusgrow medium
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