Template:Kyoto/Notebook/Sep 26

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Sep 26

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	GGA1-1
2	GGA1-2
3	GGA1-3
4	GGA1-4
5	GGA1-5
6	GGA1-6
7	GGA1-7
8	GGA1-8
9	1kbp ladder
10	GGA2-1
11	GGA2-2
12	GGA2-3
13	GGA2-4
14	GGA2-5
15	GGA2-6
16	GGA2-7
17	GGA2-8

Lane	Sample	Enzyme1	Enzyme2
1	GGA3-1
2	GGA3-2
3	GGA3-3
4	1kbp ladder
5	GGA3-4
6	GGA3-5
7	GGA3-6
8	GGA3-7

</div>

Lane	Sample	Enzyme1	Enzyme2
1	GGA3-8
2	GGA4-1
3	GGA4-2
4	GGA4-3
5	1kbp ladder
6	GGA4-4
7	GGA4-5
8	GGA4-6

</div>

Lane	Sample	Enzyme1	Enzyme2
1	GGA4-7
2	GGA4-8
3	1kbp ladder
4	GGA7-1
5	GGA16-1
6	NC(RBS-GFP-DT)

</div>

Ligasion(Golden Gate Assenbly)

No name

state	Vector		Inserter1		Inserter2		Inserter3		Inserter4		Inserter5		(NEB)T4 ligase	(NEB)10*T4 ligase	MilliQ
experiment	RBS-GFP-DT	1.0 µL	Pcon-pT181 antisense(E-2A)	0.6µL	DT(2-1)	0.5µL	Pcon-pT181 attenuator(1-SA)	2.1 µL	--	--	--	--	0.5 µL	0.5 µL	up to 5 µL
experiment	pSB1C3(EcoRI&SpeI)	1.8 µL	Pcon-pT181 antisense(E-2A)	0.9 µL	DT(2-1)	0.5 µL	Pcon-GFP-DT(1-SA)	1.5 µL	--	--	--	--	0.5 µL	0.5 µL	up to 5 µL
experiment	RBS-GFP-DT	1.0 µL	Ptet(E-0)	0.7µL	Pcon pT181 attenuator(0-1A)	0.3µL	tet aptamer(1-3)	0.3 µL	Pcon-tetR-DT(3-2A)	1.1 µL	Ptet(2-5)	0.3 µL	0.5 µL	0.5 µL	up to 5 µL
experiment	RBS-GFP-DT	1.0 µL	Ptet(E-0)	0.7 µL	Pcon-pT181 attenuator-DT(0-1A)	1.0 µL	Pcon-tetR-DT(1-2A)	1.0 µL	Ptet(2-5)	0.3µL	--	--	0.5 µL	0.5 µL	up to 5 µL
experiment	RBS-GFP-DT	1.0 µL	Pcon-pT181 attenuator-tetR-DT(E-1A)	1.0 µL	Pcon antisense-spinach-DT(1-2A)	0.7µL	Pcon-pT 181 attenuator(2-SA)	2.1 µL	--	--	--	--	0.5 µL	0.5 µL	up to 5 µL
experiment	RBS-GFP-DT	1.0 µL	Pcon antisense(E-1A)	0.5 µL	spinach-DT(1-2)	1.0 µL	Pcon-GFP-DT(1-2)	0.8 µL	--	--	--	--	0.5 µL	0.5 µL	up to 5 µL
experiment	pSB1C3(EcoRI&SpeI)	1.8 µL	Pcon-pT181 antisense(E-1A)	0.8µL	spinach-DT(1-2)	1.0µL	Pcon-GFP-DT(2-SA)	0.8 µL	--	--	--	--	0.5 µL	0.5 µL	up to 5 µL
experiment	pSB1C3(EcoRI&SpeI)	1.8 µL	Pcon-tet aptamer-DT(E-1A)	3.0 µL	Ptet-pT181 antisense(1-3)	1.5 µL	spinach-DT(3-2)	1.5 µL	Pcon-GFP-DT(2-5A)	0.8 µL	--	--	0.5 µL	0.5 µL	up to 5 µL

Liquid Culture

No name

Sample	medium
Pcon-GFP-DT	Amp
RBS-GFP-DT	CP
Pcon-pT181 attenuator-DT	Amp
Pcon-tet aptamer-DT	Amp
Pcon-spinach-DT	Amp
Pcon-pT181 antisense-spinach-DT	Amp
spinach-DT	CP

BSAI Digestion

No name

	DNA	MilliQ	BSAI-HF	10*cut smart	total
Pcon	7 µL	10.7 µL	0.3 µL	2 µL	20 µL
RBS-luxR-DT	10 µL	7 µL	0.3 µL	2 µL	20 µL

Colony PCR

Tatsui

Sample	base pair
GGA-1 (9~16)	3177
GGA-2 (9~16)	3189
GGA-3 (9~21)	2989
GGA-4 (9~16)	2939
GGA-6 (1)	3359
GGA-16(2~4)	2939

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	3min30s	30cycles

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	GGA-1 (9)	--	--
2	GGA-1 (10)	--	--
3	GGA-1 (11)	--	--
4	GGA-1 (12)	--	--
5	GGA-1 (13)	--	--
6	GGA-1 (14)	--	--
7	GGA-1 (15)	--	--
8	GGA-1 (16)	--	--
9	1kbp ladder	--	--
10	GGA-2 (9)	--	--
11	GGA-2 (10)	--	--
12	GGA-2 (11)	--	--
13	GGA-2 (12)	--	--
14	GGA-2 (13)	--	--
15	GGA-2 (14)	--	--
16	GGA-2 (15)	--	--
17	GGA-2 (16)	--	--

Lane	Sample	Enzyme1	Enzyme2
1	GGA-4 (9)	--	--
2	GGA-4 (10)	--	--
3	GGA-4 (11)	--	--
4	GGA-4 (12)	--	--
5	GGA-4 (13)	--	--
6	GGA-4 (14)	--	--
7	GGA-4 (15)	--	--
8	GGA-4 (16)	--	--
9	1kbp ladder	--	--
10	GGA-6 (1)	--	--
11	GGA-16 (2)	--	--
12	GGA-16 (3)	--	--
13	GGA-16 (4)	--	--

</div>

Lane	Sample	Enzyme1	Enzyme2
1	GGA-3 (9)	--	--
2	GGA-3 (10)	--	--
3	GGA-3 (11)	--	--
4	GGA-3 (12)	--	--
5	1kbp ladder	--	--
6	GGA-3 (13)	--	--
7	GGA-3 (15)	--	--
8	GGA-3 (14)	--	--

</div>

Lane	Sample	Enzyme1	Enzyme2
1	GGA-3 (17)	--	--
2	GGA-3 (18)	--	--
3	GGA-3 (19)	--	--
4	1kbp ladder	--	--
5	GGA-3 (20)	--	--
6	GGA-3 (21)	--	--
7	GGA-3 (16)	--	--

</div>

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	RBS-GFP-DT(RNA)	--	--
2	1kbp ladder	--	--
3	Pcon-RBS-GFP-DT(RNA)	--	--

Liquid Culture

Hirano

Sample	medium
Pcon-GFP-DT	--
RBS-GFP-DT	--
Pcon-tet aptamer-DT	--
Pcon-spinach-DT	--
Pcon-pT181 antisense-spinach-DT	--
spinach-DT	--

Colony PCR

Hirano

Sample	base pair
9/25 Pcon-pH181 attenuator+RBS-GFP-DT-1	--
9/25 Pcon-pH181 attenuator+RBS-GFP-DT-2	--
9/25 Pcon-pH181 attenuator+RBS-GFP-DT-3	--
9/25 Pcon-pH181 attenuator+RBS-GFP-DT-4	--
9/24 Ptet-RBS-GFP-DT-1	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min40s	30cycles

Ligasion(Golden Gate Assenbly)

Hirano

state	Vector	Inserter1	Inserter2	Inserter3	Inserter4	Inserter5
experiment	GGA2	Pcon-attenuator(E-1A)	Pcon-tetR-DT(1-2A)	Ptet(2-S)	RBS-GFP-DT(EcoRI&XbaI)
experiment	GGA3	Pcon-tetap-DT(E-1A)	Pcon-tetR-DT(1-2A)	Ptet(2-S)	RBS-GFP-DT(EcoRI&XbaI)
experiment	GGA4	Pcon-spi-DT(E-1A)	Pcon-tetR-DT(1-2A)	Ptet(2-S)	RBS-GFP-DT(EcoRI&XbaI)
experiment	GGA5	Pcon-antisense(E-1A)	DT(1-3)	Pcon-tetR-DT(3-2A)	Ptet(2-S)	RBS-GFP-DT(EcoRI&XbaI)
experiment	GGA6	Pcon-attenuator(E-1A)	tetap-DT(1-3)	Pcon-tetR-DT(3-2A)	Ptet(2-S)	RBS-GFP-DT(EcoRI&XbaI)
experiment	GGA7	Pcon-tetR-DT(E-2A)	Ptet(2-S)	RBS-GFP-DT(EcoRI&XbaI)

PreDenature	Denature	Annealing	Extension	cycle
37°C	16°C	50°C	80°C	--
3min	4min	5min	5min	25cycles

RNA Extraction

Nakamoto

Sample
Pcon-Spinach-DT
Pcon-tetRaptamer-DT
Pcon-attenuator-DT
Spinach-DT
Pcon-antisense-Spinach-DT

Liquid Culture

Colony PCR

Plating

Hirano

Sample	Use plate
9/26 GGA2	LB(CP)
9/26 GGA3	LB(CP)
9/26 GGA4	LB(CP)
9/26 GGA5	LB(CP)
9/26 GGA6	LB(CP)
9/26 GGA7	LB(CP)
9/26 GGA11	LB(CP)
9/26 GGA312	LB(CP)
9/26 GGA13	LB(CP)
9/26 GGA14	LB(CP)
9/26 GGA15	LB(CP)
9/26 GGA20	LB(CP)
9/26 GGA23	LB(CP)
9/26 GGA24	LB(CP)
9/26 Ptet+RBS-GFP-DT	LB(CP)

</div> incubate 37°C

Restriction Enzyme Digestion

Kojima

	9/24 pSB1C3	EcoRI	SpeI	buffer	MilliQ	total
2cuts	11µL	1µL	1µL	3µL	14µL	30µL
NC	0.5µL	0µL	0µL	1µL	8.5µL	10µL

	9/24 Pcon-pT181attenuator-aptamer12-1R-DT	EcoRI	SpeI	buffer	MilliQ	total
2cuts	8.7µL	1µL	1µL	3µL	16.3µL	30µL
NC	0.4µL	0µL	0µL	1µL	8.6µL	10µL

	9/24 pT181attenuator	EcoRI	SpeI	buffer	MilliQ	total
2cuts	3.1µL	1µL	1µL	3µL	21.9µL	30µL
NC	0.2µL	0µL	0µL	1µL	8.8µL	10µL

	8/17 RBS-GFP-DT	EcoRI	BSA	buffer	MilliQ	total
1cut	16.4µL	1µL	3µL	3µL	6.6µL	30µL
NC	0.4µL	0µL	1µL	1µL	7.6µL	10µL

	9/17 pSB4K5	EcoRI	PstI	BSA	buffer	MilliQ	total
2cuts	8.5µL	1µL	1µL	3µL	3µL	13.5µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/14 pSB4K5	EcoRI	PstI	BSA	buffer	MilliQ	total
2cuts	7.3µL	1µL	1µL	3µL	3µL	14.7µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/22 Pcon-pT181attenuator-DT	EcoRI	SpeI	buffer	MilliQ	total
2cuts	5.4µL	1µL	1µL	3µL	19.6µL	30µL
NC	0.3µL	0µL	0µL	1µL	8.7µL	10µL

	8/7 RBS-GFP-DT 1	XbaI	PstI	BSA	buffer	MilliQ	total
2cuts	8.1µL	1µL	1µL	3µL	3µL	13.9µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/26 RBS-GFP-DT 1	XbaI	BSA	buffer	MilliQ	total
1cut	8.1µL	1µL	3µL	3µL	14.9µL	30µL

	9/16 Pλ-luxI	EcoRI	PstI	BSA	buffer	MilliQ	total
2cuts	14.7µL	1µL	1µL	3µL	3µL	7.3µL	30µL
NC	0.7µL	0µL	0µL	1µL	1µL	7.3µL	10µL

	9/21 Ptet	EcoRI	SpeI	buffer	MilliQ	total
2cuts	14.2µL	1µL	1µL	3µL	10.8µL	30µL
NC	0.7µL	0µL	0µL	1µL	8.3µL	10µL

	9/24 Pcon-pT181attenuator-aptamer12-1R-DT	XbaI	PstI	BSA	buffer	MilliQ	total
2cuts	8.7µL	1µL	1µL	3µL	3µL	13.3µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

Electrophoresis

Kojima

Lane	Sample	Enzyme1	Enzyme2
1	pSB1C3	EcoRI	SpeI
2	pSB1C3	--	--
3	Pcon-pT181attenuator-aptamer12-1R-DT	EcoRI	SpeI
4	Pcon-pT181attenuator-aptamer12-1R-DT	--	--
5	1kb ladder	--	--
6	pT181attenuator	EcoRI	SpeI
7	pT181attenuator	--	--
8	RBS-GFP-DT	EcoRI	--
9	RBS-GFP-DT	--	--

Template:Kyoto/Notebook/Sep 26

From 2013.igem.org

Contents

Sep 26

Electrophoresis

Ligasion(Golden Gate Assenbly)

Liquid Culture

BSAI Digestion

Colony PCR

Electrophoresis

Electrophoresis

Liquid Culture

Colony PCR

Ligasion(Golden Gate Assenbly)

RNA Extraction

Liquid Culture

Colony PCR

Plating

Restriction Enzyme Digestion

Electrophoresis