Template:Kyoto/Notebook/Sep 22

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Sep 22

Colony PCR

Tatsui

Sample	base pair
9/20 Plac-PT181 attenuator(1~4)	664
9/20 Plac-PT181 antisense(1~6)	369
9/20 Pcon-PT181 attenuator-aptamer-DT(1~6)	859
9/20 Pcon-PT181 antisense-spinach-DT(1~8)	723

Electrophoresis

Nakamoto

Lane	Sample
1	Pcon-apt12_1R-DT(E-1A)
2	Pcon-spinach-DT(E-1A)
3	Pcon-pT181 attenuator(E-1A)
4	DT(1-3)
5	tetR aptamer 12_1R-DT(1-3)
6	100bp ladder
7	Pcon-pT181 attenuator(1-SA)
8	spinach-DT(2-1)
9	Pcon-pT181 attenuator(E-0)
10	Pcon-pT181 attenuator(2-SA)
11	spinach-DT(3-2)
12	Ptet-PT181 anisense

Miniprep

Honda

DNA	concentration[µg/mL]	260/280	260/230
9/21 spinach-DT	197.9	1.67	1.54
9/21 Plux	125.9	1.71	1.27
9/21 Plux-RBS-GFP-DT-(1)	168.0	1.62	1.73
9/21 Pcon-PT181 attenuator-(1)	368.3	1.57	1.62
9/21 RBS-GFP-DT	532.8	1.75	1.78
9/21 Pcon-RBS-GFP-DT	845.2	2.02	1.70

Colony PCR

Honda

Sample	base pair
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1~2)	283

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	2min10s	30cycles

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder	--
2~4	Pcon-aptamer12-1R-DT(E-1A)	--
6~8	Pcon-spinach-DT(E-1A)	--
10~12	Pcon-attenuator PT181(E-1A)	--

Lane	DNA	Enzyme
1	100bp ladder	--
2~4	DT(1-3)	--
6~8	aptamer12-1RDT(1-3)	--
10~12	Pcon-PT181 attenuator(1-5A)	--

Name	concentration[µg/mL]	260/280	260/230
Pcon-aptamer12-1R-DT(E-1A)	31.2	1.87	0.30
Pcon-spinach-DT(E-1A)	25.6	1.88	0.99
Pcon-attenuator PT181(E-1A)	25.3	1.99	0.86
DT(1-3)	22.1	1.82	0.20
aptamer12-1RDT(1-3)	33.5	1.99	0.05
Pcon-PT181 attenuator(1-5A)	36.9	1.12	0.96

digestion

No name

	9/16 Plac	XbaI	SpeI	PstI	BufferB	BSA	MilliQ	total
2cuts	12.5µL	0µL	1µL	1µL	3µL	3µL	9.5µL	30µL
NC	0.8µL	0µL	0µL	0µL	1µL	1µL	7.4µL	10µL

	9/22 Plux	XbaI	SpeI	PstI	BufferB	BSA	MilliQ	total
2cuts	16µL	0µL	1µL	1µL	3µL	3µL	14µL	30µL
NC	0.8µL	0µL	0µL	0µL	1µL	1µL	7.2µL	10µL

	9/20 RBS-tetR-2R-DT	XbaI	SpeI	PstI	BufferD	BSA	MilliQ	total
2cuts	8.5µL	1µL	0µL	1µL	3µL	3µL	13.5µL	30µL
NC	0.4µL	0µL	0µL	0µL	1µL	1µL	7.6µL	10µL

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder	--
2~4	Pcon-attenuator PT181(E-0)	--

Lane	DNA	Enzyme
1	100bp ladder	--
2~4	Pcon-attenuator PT181(E-0)	--

Lane	DNA	Enzyme
2	100bp ladder	--
4~6	Pcon-attenuator PT181(2-SA)	--

Lane	DNA	Enzyme
1	100bp ladder	--
3~5	spinach-DT(3-2)	--
8~10	Ptet-antisense PT181(1-3)	--

Name	concentration[µg/mL]	260/280	260/230
Pcon-attenuator PT181(E-0)	2.4	1.80	0.17
Pcon-attenuator PT181(2-SA)	10.8	1.85	0.54
spinach-DT(3-2)	36.7	1.80	0.75
Ptet-antisense PT181(1-3)	16.8	1.74	0.78

Colony PCR

No name

Sample	base pair
9/20 Pcon-PT181 attenuator-aptamer12-1R-DT-(7~14)	859

Colony PCR

Tatsui

Sample	base pair
PconPT181antisense-spinach-DT(9~15)	723

Ligation

No name

state	Vector		Inserter		Ligation High ver.2
experiment	9/13 Pcon-PT181 attenuator (SpeI & PstI)	1.7 µL	9/18 aptamer-1R-DT (XbaI & PstI)	5.4µL	3.5µL
experiment	9/12 Plac(SpeI & PstI)	2.4 µL	9/18 aptamer-1R-DT (XbaI & PstI)	6.1µL	3.5 µL
experiment	9/13 Ptet(SpeI & PstI)	1.7 µL	9/10 Plux-RBS-GFP-DT	7.0 µL	4.4 µL
experiment	9/21 RBS-GFP-DT (EcoRI & XbaI)	0.6 µL	9/18 aptamer 12-1R-DT (XbaI & PstI)	5.6µL	3.1 µL
experiment	9/13 PSB1C3(XbaI & PstI)	1.9 µL	9/16 PT181 attenuator(XbaI & PstI)	4.6µL	3.3 µL

Liquid Culture

No name

Sample	medium
9/20 Plac-antisense-1	--
9/20 Plac-attenuator-1	--
9/20 Plac-antisense-1	--
9/20 Plac-attenuator-1	--

digestion

No name

	9/8 Pcon-PT181 antisense	EcoRI	SpeI	BufferM	MilliQ	total
2cuts	8.3µL	1µL	1µL	3µL	16.7µL	30µL
NC	0.4µL	0µL	0µL	1µL	8.3µL	10µL

	9/7 Pcon-RBS-tetR-DT	EcoRI	XbaI	SpeI	PstI	BufferD	BSA	MilliQ	total
2cut	14µL	1µL	1µL	0µL	0µL	3µL	3µL	18.2µL	30µL
NC	0.7µL	0µL	0µL	0µL	0µL	1µL	1µL	7.3µL	10µL

	9/22 RBS-GFP-DT	EcoRI	XbaI	SpeI	PstI	BufferD	BSA	MilliQ	total
2cuts	3.8µL	1µL	1µL	0µL	0µL	3µL	3µL	18.2µL	30µL
NC	0.2µL	0µL	0µL	0µL	0µL	1µL	1µL	7.8µL	10µL

	9/6 aptamer12-1R-DT	EcoRI	XbaI	SpeI	PstI	BufferD	BSA	MilliQ
2cut	8µL	1µL	1µL	0µL	0µL	3µL	3µL	16µL
NC	0.4µL	0µL	0µL	0µL	0µL	1µL	1µL	7.6µL

incubate 37°C 1hour

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kbp ladder	--
3~5	Pcon-PT181 antisense (EcoRI&SpaI)	--

Transformation

No name

Name	Sample	Competent Cells	Plate
9/20 Plac-aptamer12-1R-DT(XbaI&PstI)	2 µL	20 µL	Amp
9/20 pT181 attenuator(1C3)(EcoRI&SpeI)	2 µL	20 µL	CP
9/17 pT181 attenuator(1C3)(XbaI&PstI)	2 µL	20 µL	CP
9/20 Pcon-pT181 attenuator(SpaI&PstI)＋aptamer12-1R-DT(XbaI&PstI)	2 µL	20 µL	Amp
kaiABC	2 µL	20 µL	Amp
9/22 pSB1C3-RpaB(XbaI&PstI)	2 µL	20 µL	Amp
9/22 pSB1C3-pT181 attenuator(XbaI&PstI)	2 µL	20 µL	Amp
9/22 Ptet(SpaI&PstI)＋RBS-GFP-DT(XbaI&PstI)	2 µL	20 µL	CP
9/22 RBS-GFP-DT＋Pcon-attenuator pT181 (EcoRI&SpeI)	2 µL	20 µL	CP
9/22 pT181 attenuator(XbaI&PstI)(1C3)	2 µL	20 µL	CP

PCR

No name

Sample	base pair
Pcon-RBS-tetR-DT(1-2Ⓐ)	--
Pcon-RBS-tetR-DT(3-2Ⓐ)	--
Pcon-RBS-GFP-DT(1-SⒶ)	--
Pcon-RBS-GFP-DT(2-SⒶ)	--
Pcon-tetR-DT(E-2Ⓐ)	--

PreDenature	Denature	Annealing	Extension	cycle
94 °C	94 °C	57 °C	68 °C	--
2 min	10 s	30 s	1min5s	30 cycles

Electrophoresis

No name

Lane	Sample
1	1kbp ladder
2	Pcon-RBS-tetR-DT(1-2Ⓐ)
3	Pcon-RBS-tetR-DT(3-2Ⓐ)
4	Pcon-RBS-GFP-DT(1-SⒶ)
5	Pcon-RBS-GFP-DT(2-SⒶ)
6	Pcon-tetR-DT(E-2Ⓐ)

Template:Kyoto/Notebook/Sep 22

From 2013.igem.org

Contents

Sep 22

Colony PCR

Electrophoresis

Miniprep

Colony PCR

Gel Extraction

digestion

Gel Extraction

Colony PCR

Colony PCR

Ligation

Liquid Culture

digestion

Gel Extraction

Transformation

PCR

Electrophoresis