Template:Kyoto/Notebook/Sep 24

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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)

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)

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

</div>

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
	9/22 aptamer12-1R-DT(EcoRI&XbaI)	1.4 µL	9/24 Pcon-pT181attenuator(EcoRI&SpeI)	26 µL	3.5 µL
	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

File:Igku xxxbefore.jpg
File:Igkuxxx1-2.jpg

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

File:Igkuxxxxpg
File:Igkuxxxx2.jpg

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

File:Igkuxxxx.jpg

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

File:Igkuxxxx.jpg

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

File:Igkuxxxx.jpg

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

File:Igkuxxxx.jpg

Template:Kyoto/Notebook/Sep 24

From 2013.igem.org

Contents

Sep 24

Electrophoresis

Transformation

Liquid Culture

Transformation

Liquid Culture

Digestion

Electrophoresis

Miniprep

Gel Extraction

Colony PCR

Restriction Enzyme Digestion

Ligation

colony PCR

Miniprep

Electrophoresis

Gel Extraction

Electrophoresis