Template:Kyoto/Notebook/Sep 12

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Sep 12

Miniprep

Nakamoto

DNA	concentration[µg/mL]	260/280	260/230
Pcon-pT181atte-DT	293.6	1.88	1.84
Pcon-apt12-1R-DT	303.8	1.75	1.64
Fusion1atte(pSB1c3)	172.0	1.96	2.31
RBS-lisis1-DT	235.1	1.59	1.60
RBS-lisis2-DT	387.7	1.91	2.01
RBS-lisis3-DT	387.9	1.91	2.09
Plux	166.7	1.98	1.63

Electrophoresis

Nakamoto

Lane	Sample
1	100bp ladder
2	9/11 P-RBS-lisis1-DT(Colony PCR prodution)

File:Igku xxxxxx.xxx

Liquid Culture

Nakamoto

Sample	medium
9/10 Pcon-apt12-1R-DT1	Plusgrow medium(+Amp)
9/10 DT-Pcon-pT181atte1	Plusgrow medium(+CP)
9/10 Fusion1-atte(pSB1c3)	Plusgrow medium(+CP)

Electrophoresis

Tatsui

1

Lane	Sample
1	1kb ladder
2	pSB4K5(EcoRI&SpeI)
3	pSB4K5 NC(EcoRI&SpeI)
4	RBS-lacz-DT(EcoRI&SpeI)
5	EcoR1&Spe1 NC( EcoRI&SpeI)
6	Pcon-RBS-lacz-DT( EcoRI&SpeI)
7	Pcon-RBS-lacz-DT NC( EcoRI&SpeI)
8	1kb ladder

File:Igku xxxxxx.xxx

2

Lane	Sample
1	1kb ladder
2	Ptet-pT181anti(SpeI&PstI)
3	Ptet-pT181anti NC(SpeI&PstI)
4	RBS-lysis2-DT(XbaI&PstI)
5	EcoR1&Spe1 NC( EcoRI&SpeI)
6	--
7	--
8	1kb ladder

File:Igku xxxxxx.xxx

Gel Extraction

Tatsui

Lane	DNA	Enzyme
1	1kb ladder	--
2	RBS-lacZα-DT	EcoRI&SpeI
3	RBS-lacZα-DT	EcoRI&SpeI
4	RBS-lacZα-DT	EcoRI&SpeI
5	--	--
6	Pcon-RBS-lacZα-DT	EcoRI&SpeI
7	Pcon-RBS-lacZα-DT	EcoRI&SpeI
8	Pcon-RBS-lacZα-DT	EcoRI&SpeI
9	1kb ladder	--

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Name	concentration[µg/mL]	260/280	260/230
RBS-lacZα-DT(EcoRI&SpeI)	9.8	1.50	0.39
Pcon-RBS-lacZα-DT(EcoRI&SpeI)	11.2	1.47	0.08

Gel Extraction

Tatsui

Lane	DNA	Enzyme
1	1kb ladder	--
2	RBS-lysis2-DT	XbaI&PstI
3	RBS-lysis2-DT	XbaI&PstI
4	RBS-lysis2-DT	XbaI&PstI
5	--	--
6	1kb ladder	--

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Name	concentration[µg/mL]	260/280	260/230
RBS-lysis2-DT(XbaI&PstI)	5.4	1.43	0.77

Electrophoresis

No name

Lane	Sample
1	1kb ladder
2	Pλ-RBS-luxI-DT(PCR product)
3	Pbad/araC-RBS-RFP(PCR product)

Colony PCR

Honda

Sample	base pair
9/11 apt12-1M(pSB1C3) 1	513
9/11 apt12-1M(pSB1C3) 2	513
9/11 Fusion3m2a09ttenuator(pSB1C3) 1	609
9/11 Fusion6 antisense(pSB1C3)1	431
9/11 aptamer 12-P(pSB1C3)1	515
9/11 Fusion1 antisense(pSB1C3) 1	420
9/11 Fusion1 antisense(pSB1C3) 2	420
9/11 Plac(BBa-R0011) 1	293
9/11 Plac(BBa-R0011) 2	293
9/11 Plac(BBa-R0011) 3	293
9/11 Plac(BBa-R0011) 4	293
9/11 Pcon-luxR-Plux-GFP 1	2138
9/11 Pcon-RBS-lacZα-DT(pSB4K5) 1	712

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	2min12s	30cycles

Restriction Enzyme Digestion

Tatsui

	9/5 Pcon	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
1cuts(PstI)	5.7	0µL	0µL	0µL	1µL	3µL	3µL	17.3µL	30µL
NC(PstI)	1.9	0µL	0µL	0µL	0µL	1µL	1µL	6.1µL	10µL

	8/17 DT	EcoRI	Buffer	BSA	MilliQ	total
1cut(EcoRI)	10.6	1µL	3µL	3µL	12.4µL	30µL
NC(EcoRI)	3.5	0µL	1µL	1µL	4.5µL	10µL

Colony PCR

Nakamoto,Tatsui

Sample	base pair
9/11 Pcon-pT181atte-RBS-lacZα-DT	965
9/11 Plux-PBS-lysis1-DT 1	613
9/11 Pcon-pT181attenuator-aptamer12-1R-DT 1	859
9/11 Pcon-pT181attenuator-aptamer12-1R-DT 2	859
9/11 Plux-RBS-lysis3-DT 1	1210
9/11 Plux-RBS-lysis3-DT 2	1210
Pcon-Spinach-DT(pSB4K5) 1	605

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min12s	30cycles

Transformation

tatsui

Name	Sample	Competent Cells	Plate
9/7 pSB4K5	2µL	20µL	Amp

Electrophoresis

No name

1

Lane	Sample
1	Fusion6 antisense
2	Fusion1 antisense-1
3	Fusion1 antisense-2
4	100bp ladder
5	Plac(BBa-R0011) 1
6	Plac(BBa-R0011) 2
7	Plac(BBa-R0011) 3
8	Plac(BBa-R0011) 4

File:Igku xxxxxx.xxx

2

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

Electrophoresis

No name

Lane	Sample	Enzyme
1	Pcon-pT181attenuator-RBS-lacZα-DT 1	--
2	Pcon-pT181attenuator-RBS-lacZα-DT 2	--
3	Plux-RBS-lysis1-DT 1	--
4	Pcon-pT181attenuator-aptamer12-1R-DT 1	--
5	Pcon-pT181attenuator-aptamer12-1R-DT 2	--
6	Plux-RBS-lysis3-DT 1	--
7	1kbp ladder	--
8	Plux-RBS-lysis3-DT 2	--
9	Pcon-spinach-DT(pSB4K5) 1	--
10	9/5 Pcon	PstI
11	9/5 Pcon NC	--
12	8/17 DT	EcoRI
13	8/17 DT	--

Liquid Culture

Nakamoto

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

Colony PCR

Nakamoto

Sample	base pair
9/11 Fusion1 antisense 3	394
9/11 Fusion1 antisense 4	394
9/11 aptamer12-P 2	378
9/11 aptamer12-P 3	378

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	36s	30cycles

File:Igku Aug19electrophoresis1.png

Electrophoresis

Nakamoto

Lane	Sample	Enzyme
1	Fusion1 anti 2	--
2	Fusion1 anti 3	--
3	1kb ladder	--
4	apt12-P 2	--
5	apt12-P 3	--

Restriction Enzyme Digestion

tatsui

	9/12 RBS-lysis2-DT	Xba1	PstI	Buffer	BSA	MilliQ	total
2 cuts	5.2	1.0µL	1.0µL	3.0µL	3.0µL	16.8µL	30µL
NC	0.3µL	0µL	0µL	1.0µL	1.0µL	7.7µ	10µL

	9/12 RBS-lysis3-DT	Xba1	PstI	Buffer	BSA	MilliQ	total
2 cuts	5.2	1.0µL	1.0µL	3.0µL	3.0µL	16.8µL	30µL
NC	0.3µL	0µL	0µL	1.0µL	1.0µL	7.7µ	10µL

	9/11 RBS-lysis1-DT	Xba1	PstI	Buffer	BSA	MilliQ	total
2 cuts	8.5	1.0µL	1.0µL	3.0µL	3.0µL	13.5µL	30µL
NC	0.4µL	0µL	0µL	1.0µL	1.0µL	7.6µ	10µL

	8/20 J23100-RBS-luxR -DT	EcoR1	Buffer	BSA	MilliQ	total
1cut	5.8	1.0µL	3.0µL	3.0µL	17.2µL	30µL
NC	0.3µL	0µL	1.0µL	1.0µL	7.7µ	10µL

	9/10 pSB1C3	EcoR1	Spe1	Buffer	BSA	MilliQ	total
2cuts	8.1	1.0µL	1.0µL	3.0µL	3.0µL	13.9µL	30µL
NC	0.4µL	0µL	0µL	1.0µL	1.0µL	7.6µ	10µL

	9/12 Plux	Pst1	Buffer	BSA	MilliQ	total
1cut	12.0	1.0µL	3.0µL	3.0µL	11.0micro;L	30µL
NC	0.6µL	0µL	1.0µL	1.0µL	7.4µ	10µL

	9/10 pSB1C3	Xba1	Pst1	Buffer	BSA	MilliQ	total
2cuts	8.1	1.0µL	1.0µL	3.0µL	3.0µL	13.9µL	30µL

Electrophoresis

Tatsui

Lane	Sample	Enzyme1	Enzymel2
1	9/12 RBS-lysis2-DT	Xba1	Pst1
2	9/12 RBS-lysis2-DT NC	--	--
3	9/11 RBS-lysis1-DT	Xba1	Pst1
4	9/11 RBS-lysis1-DT NC	--	--
5	9/11 RBS-lysis3-DT	Xba1	Pst1
6	9/11 RBS-lysis3-DT NC1	--	--
7	1kb ladder	--	--
8	100bp ladder	--	--
9	J23100-RBS-luxR-DT	EcoR1	--
10	J23100-RBS-luxR-DT	--	--
11	9/10 pSB1C3	EcoR1	Spe1
12	9/10 pSB1C3 NC	--	---
13	9/10 pSB1C3	Xba1	Pst1
14	9/12 Plux	Pst1	--
15	9/12 Plux NC	--	--

Template:Kyoto/Notebook/Sep 12

From 2013.igem.org

Contents

Sep 12

Miniprep

Electrophoresis

Liquid Culture

Electrophoresis

Gel Extraction

Gel Extraction

Electrophoresis

Colony PCR

Restriction Enzyme Digestion

Colony PCR

Transformation

Electrophoresis

Electrophoresis

Liquid Culture

Colony PCR

Electrophoresis

Restriction Enzyme Digestion

Electrophoresis