Template:Kyoto/Notebook/Sep 24

From 2013.igem.org

Sep 24

Electrophoresis

No name

Lane	Sample
1	9/20 Pcon-pT181aptamer12-1R-DT 1
2	9/20 Pcon-pT181aptamer12-1R-DT 2
3	9/20 Pcon-pT181aptamer12-1R-DT 3
4	9/20 Pcon-pT181aptamer12-1R-DT 4
5	9/20 Pcon-pT181aptamer12-1R-DT 5
6	9/20 Pcon-pT181aptamer12-1R-DT 6
7	9/20 Pcon-pT181aptamer12-1R-DT 7
8	9/20 Pcon-pT181aptamer12-1R-DT 8
9	100bp ladder
10	9/22 Pcon-pT181attenuator-aptamer12-1R-DT 1
11	9/22 Pcon-pT181attenuator-aptamer12-1R-DT 2
12	9/22 Pcon-pT181attenuator-aptamer12-1R-DT 3
13	9/22 Pcon-pT181attenuator-aptamer12-1R-DT 4
14	9/20 Plac-aptamer12-1R-DT 1
15	9/20 Plac-aptamer12-1R-DT 2
16	9/20 Plac-aptamer12-1R-DT 3
17	9/20 Plac-aptamer12-1R-DT 4

Transformation

No name

Name	Sample	Competent Cells	Total	Plate
9/23 pSB1C3-pT181attenuator	2 µL	20 µL	22 µL	LB (+CP)
RBS-GFP-DT+Pcon-pT181attenuator	2 µL	20 µL	22 µL	LB (+CP)
9/23 Plac-+spinach-DT	2 µL	20 µL	22 µL	LB (+Amp)
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT	2 µL	20 µL	22 µL	LB (+Amp)
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT	2 µL	20 µL	22 µL	LB (+Amp)
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT	2 µL	20 µL	22 µL	LB (+Amp)
9/23 Pcon-pT181antisense-DT	2 µL	20 µL	22 µL	LB (+CP)
9/23 Pcon-pT181attenuator-RBS-GFP-DT	2 µL	20 µL	22 µL	LB (+CP)
9/23 Pcon-pT181attenuator-aptamer12-1R-DT	2 µL	20 µL	22 µL	LB (+CP)
PUC19	2 µL	20 µL	22 µL	LB (+Amp)
Ptet-RBS-lacZα-DT	2 µL	20 µL	22 µL	LB (+CP)
pSB1C3	2 µL	20 µL	22 µL	LB (+CP)

Liquid Culture

No name

Sample	medium
aptamer12-1R-DT(Master plate)	LB(+CP)

Restriction Enzyme Digestion(PCR product)

Hirano

Concentrate PCR product by evaporator until volume become less than 1µL, add all sulution and pipetting.

	10*cut smart buffer	BsaI-HF	MilliQ	total
9/22 Pcon-pT181 attenuator (E-0)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Pcon-pT181 attenuator (2-3A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 spinach-DT(3-2)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Ptet-pT181 antisense (1-3)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Pcon-tet aptamer-DT (E-1A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Pcon-spinach-DT (E-1A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Pcon-pT181 attenuator (E-1A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 DT (1-3)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 tet aptamer-DT (1-3)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Pcon-pT181 attenuator (1-SA)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Pcon-RBS-tetR-DT (1-2A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Pcon-RBS-tetR-DT (3-2A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Pcon-RBS-tetR-DT (E-2A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Pcon-RBS-GFP-DT (1-2A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Pcon-RBS-GFP-DT (2-SA)	2.0 µL	0.3 µL	17 µL	19.3µL
9/23 Pcon-pT181 attenuator-DT (E-1A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/23 Pcon-pT181 attenuator-DT (0-1A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/23 Pcon-pT181 antisense-spinach-DT (1-2A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/23 Pcon-pT181 antisense (1-2A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/23 Ptet-1 (2-S)	2.0 µL	0.3 µL	17 µL	19.3µL
9/23 Plac-2 (E-1A)	2.0 µL	0.3 µL	17 µL	19.3µL

Incubate 37°C 12hour</div>

Column Refining

Kojima

Name	concentration[µg/mL]	260/280	260/230
8/22 Pcon-pT181 attenuator(E-0)	7.8	1.70	0.03
8/22 Pcon-pT181 attenuator(2-SA)	17.3	1.93	0.03
8/22 spinach-DT (3-2)	22.4	1.93	0.09
8/22 Ptet-PT181 anisense (1-3)	18.3	1.58	0.14
8/22 Pcon-aptamer12_1R-DT (E-1A)	8.2	2.10	0.05
8/22 Pcon-spinach-DT(E-1A)	5.6	0.91	0.05
8/22 Pcon-pT181 attenuator(E-1A)	18.5	1.80	0.12
8/22 DT(1-3)	7.3	1.13	0.04
8/22 tetR aptamer 12_1R-DT(1-3)	14.6	1.74	0.06
8/22 Pcon-pT181 attenuator(1-SA)	7.9	1.57	0.04
8/22 Pcon-RBS-tetR-DT(1-2Ⓐ)	41.6	1.81	0.25
8/22 Pcon-RBS-tetR-DT(3-2Ⓐ)	30.2	1.75	0.15
8/22 Pcon-RBS-tetR-DT(E-2Ⓐ)	36.9	1.75	0.56
8/22 Pcon-RBS-GFP-DT(1-SⒶ)	43.5	1.83	0.33
8/22 Pcon-RBS-GFP-DT(2-SⒶ)	64.6	1.82	0.30
8/23 Pcon-pT181 attenuator-DT (E-1A)	31.2	1.83	0.09
8/23 Pcon-pT181 attenuator-DT (0-1A)	64.5	1.80	0.43
8/23 Pcon-pT181 antisense-spinach-DT (1-2A)	36.4	1.88	0.21
8/23 Pcon-pT181 antisense-spinach-DT (E-1A)	27.9	1.92	0.16
8/23 Ptet(1)(2-S)	37.0	1.91	0.16
9/23 Plac-2(E-1A)	21.2	1.82	0.13

Transformation

No name

Name	Sample	Competent Cells	Total	Plate
9/23 Pcon-attenuator-aptamer12-1R-DT	2 µL	20 µL	22 µL	LB (+CP)
9/23 pT181attenuator(1C3)	2 µL	20 µL	22 µL	LB (+CP)
9/23 Pcon-pT181antisense-DT	2 µL	20 µL	22 µL	LB (+CP)
9/23 Pcon-pT181attenuator-RBS--GFP-DT	2 µL	20 µL	22 µL	LB (+CP)

Liquid Culture

No name

Sample	medium
pSB1C3 (Master plate-1)	LB(+CP)

Restriction Enzyme Digestion

Kojima

	8/21 pT181attenuator (330 µg/mL)	XbaI	PstI	buffer	BSA	MilliQ	total
2 cuts	6.1	1	1	3	3	15.9	30
NC	0.3	0	0	1	1	7.7	10

	9/22 Pcon-pT181attenuator (368.3 µg/mL)	EcoRI	SpeI	buffer	BSA	MilliQ	total
2 cuts	5.4	1	1	3	0	19.6	30
NC	0.3	0	0	1	0	8.7	10

	9/16 pSB1C3	EcoRI	SpeI	buffer	BSA	MilliQ	total
2 cuts	18	1	1	3	0	7.0	30
NC	0.9	0	0	1	0	8.1	10

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	1kb ladder	-	-
2	pT181attenuator	XbaI	PstI
3	pT181attenuator	--	--
4	Pcon-pT181attenuator	EcoRI	SpeI
5	Pcon-pT181attenuator	--	--
6	pSB1C3	EcoRI	SpeI
7	pSB1C3	--	--

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
9/24 aptamer12-1R-DT	144.7	1.03	1.21

Gel Extraction

Kojima

Lane	DNA	Enzyme1	Enzyme2
1	100kb ladder	--	--
2	pT181attenuator	XbaI	PstI
3	pT181attenuator	XbaI	PstI
4	pT181attenuator	XbaI	PstI
5	--	--	--
6	Pcon-pT181attenuator	EcoRI	SpeI
7	Pcon-pT181attenuator	EcoRI	SpeI
8	Pcon-pT181attenuator	EcoRI	SpeI
9	--	--	--
10	pSB1C3	EcoRI	SpeI
11	pSB1C3	EcoRI	SpeI
12	pSB1C3	EcoRI	SpeI

Name	concentration[µg/mL]	260/280	260/230
pT181attenuator(XbaI&PstI)	10.1	2.02	0.43
Pcon-pT181attenuator(EcoRI&SpeI)	10.3	1.81	0.32
pSB1C3(EcoRI&SpeI)	10.0	2.81	0.02

Colony PCR

Nakamoto

Sample	base pair
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 1	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 2	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 3	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 4	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 5	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 6	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 7	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 8	1414
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 1	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 2	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 3	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 4	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 5	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 6	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 7	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 8	1631
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 1	2042
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 2	2042
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 3	2042
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 4	2042
9/23 Pcon-pT181antisense-DT 1	508
9/23 Pcon-pT181antisense-DT 2	508
9/23 Pcon-pT181antisense-DT 3	508
9/23 Pcon-pT181antisense-DT 4	508
9/23 Pcon-pT181antisense-DT 5	508
9/23 Pcon-pT181antisense-DT 6	508
9/23 Pcon-pT181antisense-DT 7	508
9/23 Pcon-pT181antisense-DT 8	508

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

Lane	Sample
1	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 1
2	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 2
3	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 3
4	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 4
5	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 5
6	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 6
7	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 7
8	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 8
9	1kb ladder
10	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 1
11	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 2
12	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 3
13	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 4
14	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 5
15	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 6
16	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 7
17	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 8

Lane	Sample
1	9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 1
2	9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 2
3	9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 3
4	9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 4
5	9/23 Pcon-pT181antisense-DT 1
6	9/23 Pcon-pT181antisense-DT 2
7	1kb ladder
8	9/23 Pcon-pT181antisense-DT 3
9	9/23 Pcon-pT181antisense-DT 4
10	9/23 Pcon-pT181antisense-DT 5
11	9/23 Pcon-pT181antisense-DT 6
12	9/23 Pcon-pT181antisense-DT 7
13	9/23 Pcon-pT181antisense-DT 8
14	--

</div>

Restriction Enzyme Digestion

Nakamoto

	9/24 aptamer12-1R-DT	EcoRI	XbaI	buffer	BSA	MilliQ	total
2cuts	17.5µL	1.0µL	1.0µL	3.0µL	3.0µL	4.5µL	30µL
NC	0.9µL	0µL	0µL	1.0µL	1.0µL	7.1µL	10µL

	9/20 Ptet	SpeI	PstI	buffer	BSA	MilliQ	total
2cuts	9.5µL	1.0µL	1.0µL	3.0µL	0µL	15.5µL	30µL
NC	0.5µL	0µL	0µL	1.0µL	0µL	8.5µL	10µL

	9/22 RBS-GFP-DT	EcoRI	SpeI	buffer	BSA	MilliQ	total
2cuts	3.8µL	1.0µL	1.0µL	3.0µL	0µL	21.2µL	30µL
NC	0.2µL	0µL	0µL	1.0µL	0µL	8.8µL	10µL

	9/22 Pcon-RBS-GFP-DT	EcoRI	XbaI	buffer	BSA	MilliQ	total
2cuts	2.4µL	1.0µL	1.0µL	3.0µL	3.0µL	19.6µL	30µL
NC	0.1µL	0µL	0µL	1.0µL	1.0µL	7.9µL	10µL

	9/22 Pcon-pT181attenuator 1	EcoRI	XbaI	buffer	BSA	MilliQ	total
2cuts	5.4µL	1.0µL	1.0µL	3.0µL	3.0µL	16.6µL	30µL
NC	0.3µL	0µL	0µL	1.0µL	1.0µL	7.7µL	10µL

	9/22 RBS-GFP-DT	XbaI	PstI	buffer	BSA	MilliQ	total
2cuts	3.8µL	1.0µL	1.0µL	3.0µL	3.0µL	18.2µL	30µL
NC	0.2µL	0µL	0µL	1.0µL	1.0µL	7.8µL	10µL

Ligation

Nakamoto

state	Vector		Inserter		Ligation High ver.2
experiment	9/22 aptamer12-1R-DT(EcoRI&XbaI)	1.4 µL	9/24 Pcon-pT181attenuator(EcoRI&SpeI)	26 µL	3.5 µL
experiment	9/4 Ptet(SpeI&PstI)	2.0 µL	9/21 RBS-GFP-DT(XbaI&PstI)	9.0 µL	3.5 µL

incubated at 37 °C for 1 hour.

colony PCR

Kojima

Sample	base pair
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 5	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 6	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 7	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 8	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 9	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 10	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 11	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 12	2042bp
9/23 Pcon-pT181antisense-DT 9	508bp
9/23 Pcon-pT181antisense-DT 10	508bp
9/23 Pcon-pT181antisense-DT 11	508bp
9/23 Pcon-pT181antisense-DT 12	508bp
9/23 Pcon-pT181antisense-DT 13	508bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 1	1527bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 2	1527bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 3	1527bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 4	1527bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 1	859bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 2	859bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 3	859bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 4	859bp
9/24 pT181attenuator(pSB1C3) 1	390bp
9/24 pT181attenuator(pSB1C3) 2	390bp
9/24 pT181attenuator(pSB1C3) 3	390bp
9/24 pT181attenuator(pSB1C3) 4	390bp

Tatsui

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

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
pSB1C3	183.9	1.91	1.59
Pcon-pT181attenuator-aptamer12-1R-DT	231.7	1.82	2.35
Plac-aptamer12-1R-DT	208.9	1.98	1.79

Electrophoresis

Nakamoto

Lane	Sample	Enzyme1	Enzyme2
1	1kb ladder	--	--
2	9/24 aptamer12-1R-DT	EcoRI	XbaI
3	9/24 aptamer12-1R-DT	--	--
4	9/24 Ptet	SpeI	PstI
5	9/24 Ptet	--	--
6	9/24 RBS-GFP-DT	EcoRI	SpeI
7	9/24 RBS-GFP-DT	--	--
8	9/24 Pcon-RBS-GFP-DT	EcoRI	XbaI
8	9/24 Pcon-RBS-GFP-DT	--	--
9	9/24 Pcon-RBS-GFP-DT	--	--
10	9/24 Pcon-pT181attenuator 1	EcoRI	XbaI
11	9/24 Pcon-pT181attenuator 1	--	--
12	9/24 RBS-GFP-DT	XbaI	PstI
13	9/24 RBS-GFP-DT	--	--
14	1kb ladder	--	--

Gel Extraction

No name 1

Lane	DNA	Enzyme1	Enzyme2
1	1kb ladder	--	--
2	aptamer12-1R-DT	EcoRI	XbaI
3	aptamer12-1R-DT	EcoRI	XbaI
4	aptamer12-1R-DT	EcoRI	XbaI
5	--	--	--
6	Ptet	SpeI	PstI
7	Ptet	SpeI	PstI
8	Ptet	SpeI	PstI
9	--	--	--
10	RBS-GFP-DT	EcoRI	SpeI
11	RBS-GFP-DT	EcoRI	SpeI
12	RBS-GFP-DT	EcoRI	SpeI

Lane	DNA	Enzyme1	Enzyme2
1	1kb ladder	--	--
2	Pcon-RBS-GFP-DT	EcoRI	XbaI
3	Pcon-RBS-GFP-DT	EcoRI	XbaI
4	Pcon-RBS-GFP-DT	EcoRI	XbaI
5	--	--	--
6	Pcon-pT181attenuator	EcoRI	XbaI
7	Pcon-pT181attenuator	EcoRI	XbaI
8	Pcon-pT181attenuator	EcoRI	XbaI
9	--	--	--
10	RBS-GFP-DT	XbaI	PstI
11	RBS-GFP-DT	XbaI	PstI
12	RBS-GFP-DT	XbaI	PstI

Electrophoresis

No name
1

Lane	Sample
1	1kb ladder
2	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 5
3	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 6
4	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 7
5	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 8
6	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 9
7	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 10
8	1kb ladder

2

Lane	Sample
1	1kb ladder
2	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 11
3	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 12
4	Pcon-pT181attenuator-RBS-GFP-DT 1
5	Pcon-pT181attenuator-RBS-GFP-DT 2
6	Pcon-pT181attenuator-RBS-GFP-DT 3
7	Pcon-pT181attenuator-RBS-GFP-DT 4
8	1kb ladder

3

Lane	Sample
1	Pcon-pT181antisense-DT 9
2	Pcon-pT181antisense-DT 10
3	Pcon-pT181antisense-DT 11
4	Pcon-pT181antisense-DT 12
5	Pcon-pT181antisense-DT 13
6	100bp ladder
7	Pcon-pT181attenuator-aptamer12-1R-DT 1
8	Pcon-pT181attenuator-aptamer12-1R-DT 2

4

Lane	Sample
1	Pcon-pT181attenuator-aptamer12-1R-DT 3
2	Pcon-pT181attenuator-aptamer12-1R-DT 4
3	100bp ladder
4	pT181attenuator(pSB1C3) 1
5	pT181attenuator(pSB1C3) 2
6	pT181attenuator(pSB1C3) 3
7	pT181attenuator(pSB1C3) 4

Ligation

Hirano

state	Vector		Inserter1		Inserter2		Inserter3		Inserter4		(NEB)T4ligase	(NEB)Buffer	milliQ	total
experiment	RBS-GFP-DT(pSB1C3)	1µL	tetR-aptamer12-1R-DT(1-3)	1 µL	Ptet(2-S)	0.3µL	Pcon-tetR-aptamer12-1R-DT(3-2A)	1.1µL	Plac(E-1A)	0.5µL	0.5µL	0.5µL	0.2 µL	5µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-pT181attenuator(E-1A)	1 µL	Pcon-tetR-aptamer12-1R-DT(1-2A)	1µL	Ptet(2-S)	0.3µL	--	--	0.5µL	0.5µL	0.7 µL	5µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-tetRaptamer12-1R-DT(E-1A)	2µL	Pcon-tetRaptamer12-1R-DT(1-2A)	1µL	Ptet(2-S)	0.3µL	--	--	0.5µL	0.5µL	0 µL	5.3µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-spinach-DT(E-1A)	3µL	Pcon-tetRaptamer12-1R-DT(1-2A)	1µL	Ptet(2-S)	0.3µL	--	--	0.5µL	0.5µL	0 µL	6.3µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-pT181antisense-DT(E-1A)	0.5µL	DT(1-3)	2µL	Ptet(2-S)	0.3µL	Pcon-tetRaptamer12-1R-DT(3-2A)	1.1µL	0.5µL	0.5µL	0.1 µL	5µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-pT181attenuator(E-1A)	1µL	tetRaptamer12-1R-DT(1-3)	1µL	Ptet(2-S)	0.3µL	Pcon-tetRaptamer12-1R-DT(3-2A)	1.1µL	0.5µL	0.5µL	0µL	5.4µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Ptet(2-S)	0.3µL	Pcon-tetRaptamer12-1R-DT(E-2A)	1µL	--	--	--	--	0.5µL	0.5µL	1.7µL	5µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-GFP-DT(2-SA)	0.5µL	1µL	--	--	--	--	--	0.5µL	0.5µL	1.5µL	5µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-tetRaptamer12-1R-DT(E-1A)	2µL	Pcon-pT181attenuator(1-SA)	2.1µL	--	--	--	--	0.5µL	0.5µL	0µL	6.1µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-pT181attenuator-DT(E-1A)	1µL	Pcon-pT181attenuator(1-SA)	2.1µL	--	--	--	--	0.5µL	0.5µL	0µL	5.1µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-tetR12-1R-DT(E-1A)	2µL	Pcon-GFP-DT(2-SA)	0.5µL	spinach-DT(3-2)	1µL	Ptet-pT181antisense(1-3)	1µL	0.5µL	0.5µL	0µL	6.5µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-tetRaptamer12-1R-DT(E-1A)	2µL	Pcon-pT181antisense(1-2A)	0.7µL	Pcon-pT181attenuator(2-SA)	2.1µL	--	--	0.5µL	0.5µL	0µL	6.8µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-tetRaptamer12-1R-DT(E-1A)	2µL	Pcon-pT181attenuator(2-SA)	2.1µL	Spinach-DT(3-2)	1µL	Ptet-pT181antisense(1-3)	1µL	0.5µL	0.5µL	0µL	8.1µL

Concentrate total (more than 5µL) by evaporater until volume become less than 5 µL

PCR

No name

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	F primer	R primer	KOD-plus	MilliQ	total
Pcon antisense(1C3-GGE-fwd,1A2-GG2-rev)	1µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	0.5µL	15.5	25µL
DT-(1C3-GG1-fwd,1C3-GG2-rev)	1µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	0.5µL	15.5	25µL
Spinach-DT-(1C3-GG1-fwd,1C3-GG2-rev)	1µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	0.5µL	15.5	25µL
Pcon-antisense-spinach-DT(1C3-GGE-fwd,1A2-GG1-rev)	1µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	0.5µL	15.5	25µL
Ptet(1C3-GGE-fwd,1C3-GG0-rev)	1µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	0.5µL	15.5	25µL
Pcon attenuator(1C3-GGE-fwd,1C3-GG0-rev)	1µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	0.5µL	15.5	25µL
DT(1C3-GG2-fwd,1C3-GG1-rev)	1µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	0.5µL	15.5	25µL

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10sec	30sec	36sec	30

Template:Kyoto/Notebook/Sep 24

From 2013.igem.org

Contents

Sep 24

Electrophoresis

Transformation

Liquid Culture

Restriction Enzyme Digestion(PCR product)

Column Refining

Transformation

Liquid Culture

Restriction Enzyme Digestion

Electrophoresis

Miniprep

Gel Extraction

Colony PCR

Restriction Enzyme Digestion

Ligation

colony PCR

Miniprep

Electrophoresis

Gel Extraction

Electrophoresis

Ligation

PCR