Template:Kyoto/Notebook/Sep 5

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Sep 5

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	J23100-3	--	--
3	J23100-4	--	--
4	J23100-5	--	--
5	100bp ladder	--	--

Miniprep

Nakamoto

DNA	concentration[µg/mL]	260/280	260/230
8/3 J23100-3	349.1	1.69	1.851
8/10 Plac-2	331.3	1.84	1.42

Ligation

Kojima

state	Vector		Inserter		Ligation High ver.2
experiment	Pconst	13.2	spinach-DT	2.1	3.5
experiment	Plac	15.3	spinach-DT	2.1	3.5
experiment	Pconst	13.2	pT181 antisense (XbaI & PstI)	6.6	3.5
experiment	Plac	15.3	pT181 antisense (XbaI & PstI)	6.6	3.5
experiment	Ptet	30.1	pT181 antisense (XbaI & PstI)	6.6	3.5
experiment	Pconst	13.2	pT181 attenuator	11.7	3.5
experiment	Plac	15.3	pT181 attenuator	11.7	3.5
experiment	Ptet	30.1	RBS-lacZα-DT	3.0	3.5

incubate 16 °C 1 hour

Electrophoresis

Tatsui

Lane	Sample
1	100bp ladder
2	RBS-luxI-DT
3	Pbad/araC
4	NC
5	100bp ladder

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kbp ladder
2	RBS-luxI-DT
3	RBS-luxI-DT
4
5	Pbad/araC
6	Pbad/araC

Name	concentration[µg/mL]	260/280	260/230
RBS-luxI-DT	23.2	0.83	7.89
Pbad/araC	51.8	1.20	0.11

Colony PCR

No name

Sample	base pair
9/4 aptamer 12_1R(pSB1C3)-1	384
9/4 aptamer 12_1R(pSB1C3)-2	384
9/4 pT181 antisense(pSB1C3)-1	415
9/4 pT181 antisense(pSB1C3)-2	415
9/4 pT181 antisense(pSB1C3)-3	415
9/4 pT181 antisense(pSB1C3)-4	415

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

Sample	base pair
9/4 aptamer 12_1R-DT-1	521
9/4 aptamer 12_1R-DT-2	521
9/4 aptamer 12_1R-DT-3	521
9/4 aptamer 12_1R-DT-4	521
9/4 pT181 attenuator(pSB1C3)	601
9/4 pT181 attenuator(pSB1C3)	601
9/4 RBS-lysis1-DT	613
9/4 RBS-lysis1-DT	613

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

Sample	base pair
9/4 RBS-lysis2-DT-1	985
9/4 RBS-lysis2-DT-2	985
9/4 RBS-lysis2-DT-3	985
9/4 RBS-lysis2-DT-4	985
9/4 RBS-lysis3-DT-1	1210
9/4 RBS-lysis3-DT-2	1210

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	72s	30cycles

Liquid Culture

Nakamoto

Sample	medium
8/9 J23100-5	Plusgrow medium(+Amp)

37°C

Transformation

No name

Name	Sample(µL)	Competent Cells(µL)	Total(µL)	Plate
9/5 Pconst+spinach-DT	2	20	22	--
9/5 Plac+spinach-DT	2	20	22	--
9/5 Pconst+pT181 antisense	2	20	22	--
9/5 Plac+pT181 antisense	2	20	22	--
9/5 Ptet+pT181 antisense	2	20	22	--
9/5 Plux+pT181 attenuator	2	20	22	--
9/5 Ptet+pT181 attenuator	2	20	22	--
9/5 Ptet+pT181 attenuator	2	20	22	--
9/5 Pconst+RBS-tetR+DT	2	20	22	--
9/16(2012) T7-His-FT	2	20	22	--
9/16(2012) pBr322	2	20	22	--

Restriction Enzyme Digestion

No name

	9/5 Pbad/araC	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	4µL	0µL	1µL	0µL	1µL	3µL	3µL	18µL	30µL

	9/5 RBS-luxI-DT	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	4µL	0µL	0µL	1µL	1µL	3µL	3µL	18µL	30µL

	8/28 Plux	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	12µL	0µL	1µL	0µL	1µL	3µL	3µL	10µL	30µL
NC	0.6µL	0µL	0µL	0µL	0µL	1µL	1µL	7.4µL	10µL

	9/5 Pconst(J23100)	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	5.7µL	0µL	1µL	0µL	1µL	3µL	3µL	16.3µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	9/5 Plac	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	6.0µL	0µL	1µL	0µL	1µL	3µL	3µL	16µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	8/31 Plux-RBS-GFP-DT-1	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	9.4µL	1µL	1µL	0µL	0µL	3µL	3µL	12.6µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL

	8/20 Pconst-RBS-luxR-DT	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	4.5µL	1µL	0µL	1µL	0µL	3µL	3µL	13.5µL	30µL
NC	0.2µL	0µL	0µL	0µL	0µL	1µL	1µL	7.8µL	10µL

DNA	concentration[µg/mL]	260/280	260/230
9/5 RBS-luxI-DT	7.6	1.56	-1.42
9/5 Pbad/araC	7.5	1.54	3.84

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	aptamer 12_1R (pSB1C3)-1	--	--
3	aptamer 12_1R (pSB1C3)-2	--	--
4	pT181 antisense(pSB1C3)-1	--	--
5	pT181 antisense(pSB1C3)-2	--	--
6	pT181 antisense(pSB1C3)-3	--	--
7	pT181 antisense(pSB1C3)-4	--	--
8	100bp ladder	--	--

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	aptamer 12_1R-DT-1	--	--
3	aptamer 12_1R-DT-2	--	--
4	aptamer 12_1R-DT-3	--	--
5	aptamer 12_1R-DT-4	--	--
6	pT181 attenuator(pSB1C3)-1	--	--
7	pT181 attenuator(pSB1C3)-2	--	--
8	RBS-lysis1-DT-1	--	--
9	RBS-lysis1-DT-2	--	--
10	100bp ladder	--	--

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	RBS-lysis2-DT-1	--	--
3	RBS-lysis2-DT-2	--	--
4	RBS-lysis2-DT-3	--	--
5	RBS-lysis2-DT-4	--	--
6	RBS-lysis3-DT-1	--	--
7	RBS-lysis3-DT-2	--	--
8	100bp ladder	--	--

Lane	Sample	Enzyme1	Enzyme2
1	1kbp ladder	--	--
2	Pconst	SpeI	PstI
3	Pconst NC	--	--
4	Plac	SpeI	PstI
5	Plac NC	--	--
6	Plux	SpeI	PstI
7	Plux NC	--	--
8	Pconst-RBS-luxR-DT	EcoRI	XbaI
9	Pconst-RBS-luxR-DT NC	--	--
10	Plux-RBS-GFP-DT	EcoRI	SpeI
11	Plux-RBS-GFP-DT NC	--	--
12	1kbp ladder	--	--

5x M9 Medium (+EDTA)

Hirano

volume	10ml
Na2HPO4	60mg
KH2PO4	30mg
NaCl	5mg
NH4Cl	10mg
Fe(III)-EDTA	1263.27mg
MilliQ	up to 10 mL

autoclave at 121 °C for 20 min

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kbp ladder
2	Pconst	SpeI & PstI
3	Pconst	SpeI & PstI

Name	concentration[µg/mL]	260/280	260/230
Pconst	5.3	1.98	0.06

Lane	DNA	Enzyme
1	100bp ladder
2	Plac	SpeI & PstI
3	Plac	SpeI & PstI
5	Plux	SpeI & PstI
6	Plux	SpeI & PstI

Name	concentration[µg/mL]	260/280	260/230
Plac (SpeI & PstI)	5.2	1.82	0.36
Plux(SpeI & PstI)	8.0	1.87	0.25

Lane	DNA	Enzyme
1	1kbp ladder
2	Pconst-RBS-luxR-DT	EcoRI & XbaI
3	Pconst-RBS-luxR-DT	EcoRI & XbaI
5	Plux-RBS-GFP-DT	EcoRI & SpeI
6	Plux-RBS-GFP-DT	EcoRI & SpeI

Name	concentration[µg/mL]	260/280	260/230
Pconst-RBS-luxR-DT(EcoRI & XbaI)	30.6	1.84	1.16
Plux-RBS-GFP-DT (EcoRI & SpeI)	21.8	1.87	0.98

DNA Purification

No name

DNA	concentration[µg/mL]	260/280	260/230
9/5 RBS-luxI-DT	7.6	1.56	-1.42
9/5 Pbad/araC	7.5	1.54	3.84

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
8/9 J23100-5	243.0	1.90	1.81

Colony PCR

No name

Sample	base pair
9/4 RBS-lysis3-DT-3	1210
9/4 RBS-lysis3-DT-4	1210
9/4 RBS-lysis3-DT-5	1210
9/4 RBS-lysis1-DT-3	613
9/4 Ptrc KaiC-1	--
9/4 pSB4K5	1370

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min 25s	30cycles

Liquid Culture

Hirano

Sample	medium
9/4 aptamer 12_1R-DT-1	Plusgrow medium (+CP)
9/4 pT181 attenuator(pSB1C3)-1	Plusgrow medium (+CP)
9/4 RBS-lysis2-DT-1	Plusgrow medium (+CP)
9/4 Ptrc-KaiC -1	Plusgrow medium (+Amp)
9/4 pSB4K5 -1	Plusgrow medium (+Kan)

Template:Kyoto/Notebook/Sep 5

From 2013.igem.org

Contents

Sep 5

Electrophoresis

Miniprep

Ligation

Electrophoresis

Gel Extraction

Colony PCR

Liquid Culture

Transformation

Restriction Enzyme Digestion

Electrophoresis

5x M9 Medium (+EDTA)

Gel Extraction

DNA Purification

Miniprep

Colony PCR

Liquid Culture