Template:Kyoto/Notebook/Aug 27

From 2013.igem.org

Aug 27

Restriction Enzyme Digestion

No name

	8/21 pT181 attenuator-1(330µg/mL)	EcoRI	SpeI	10x BufferB	100x BSA	MilliQ	total
2 cuts	3.0µL	0.5µL	0.5µL	3.0µL	0.3µL	22.7µL	30µL
NC	0.6µL	0µL	0µL	1µL	0.1µL	8.3µL	10µL

	8/17 DT-1(188µg/mL)	EcoRI	XbaI	10x BufferB	100x BSA	MilliQ	total
2 cuts	3.0µL	0.5µL	0.5µL	3.0µL	0.3µL	22.7µL	30µL
NC	1.0µl	0µL	0µL	1µL	0.1µL	7.9µL	10µL

	8/20 Pcon-RBS-luxR-DT-2(344µg/mL)	EcoRI	SpeI	10x BufferB	100x BSA	MilliQ	total
2 cuts	2.9µL	0.5µL	0.5µL	3.0µL	0.3µL	22.8µL	30µL
NC	0.6µl	0µL	0µL	1µL	0.1µL	8.3µL	10µL

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	8/21 pT181 attenuator	EcoI	SpeI
3	8/21 pT181 atteniator	--	--
4	8/17 DT-1	EcoRI	XbaI
5	8/17 DT-1	--	--
6	8/20 Pcon-RBS-luxR-DT-2	EcoI	SpeI
7	8/20 Pcon-RBS-luxR-DT-2	--	--
8	100bp ladder	--	--

Colony PCR

Kojima and Yoshida

Sample	base pair
8/26 Pλ-luxI(1)
8/26 Pλ-luxI(2)
NC

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

Lane	Sample
1	100bp ladder
2	Pλ-luxI-(1)
3	Pλ-luxI-(2)
4	NC

Liquid Culture

No name

Sample	medium
8/26 Pλ-RBS-luxI-DT-1	Plusgrow medium (+Amp)
8/26 Pλ-RBS-luxI-DT-2	Plusgrow medium (+Amp)

Restriction Enzyme Digestion

No name

	8/21 pT181 attenuator-1(330µg/mL)	EcoRI	SpeI	10x BufferB	100x BSA	MilliQ	total
2 cuts	3.0µL	0.5µL	0.5µL	3.0µL	0.3µL	22.7µL	30µL
NC	0.6µL	0µL	0µL	1µL	0.1µL	8.3µL	10µL

	8/17 DT-1(188µg/mL)	EcoRI	XbaI	10x BufferB	100x BSA	MilliQ	total
2 cuts	3.0µL	0.5µL	0.5µL	3.0µL	0.3µL	22.7µL	30µL
NC	1.0µl	0µL	0µL	1µL	0.1µL	7.9µL	10µL

	8/20 Pcon-RBS-luxR-DT-2(344µg/mL)	EcoRI	SpeI	10x BufferB	100x BSA	MilliQ	total
2 cuts	2.9µL	0.5µL	0.5µL	3.0µL	0.3µL	22.8µL	30µL
NC	0.6µl	0µL	0µL	1µL	0.1µL	8.3µL	10µL

Electrophoresis

Hirano

Lane	Sample	Enzyme1	Enzyme2
	100bp ladder	--	--
1	pT181 attenuator	EcoRI	SpeI
2	pT181 attenuator	--	--
3	DT-(1)	EcoRI	XbaI
4	DT-(1)	--	--
5	Pcon-RBS-luxR-DT-(2)	EcoRI	SpeI
6	Pcon-RBS-luxR-DT-(2)	--	--
	100bp ladder	--	--

Gel Extraction

Inoue

Lane	DNA	Enzyme
1	100bpladder	--
2	pT181 attenuator-1	EcoRI&SpeI
3	pT181 attenuator-1	EcoRI&SpeI
4	DT-1	EcoRI&XbaI
5	DT-1	EcoRI&XbaI
6	Pcon-RBS-luxR-DT-2	EcoRI&SpeI
7	Pcon-RBS-luxR-DT-2	EcoRI&SpeI
8	100bpladder	--

Name	concentration[µg/mL]	260/280	260/230
pT181 attenuator-1(EcoRI&SpeI)	4.2	1.62	0.29
DT-1(EcoRI&XbaI)	17	1.88	0.88

Transformation

Kojima and Hirano

Name	Sample	Competent Cells	Total	Plate
8/26 tetR aptamer 12_1R-DT	1µL	10µL	11µL	CP
8/26 pT181 attenuator-DT	1µL	10µL	11µL	CP
8/26 pT181 antisense-DT	1µL	10µL	11µL	CP
8/26 Spinach-DT	1µL	10µL	11µL	CP
8/26 Plac-pT181 attenuator	1µL	10µL	11µL	CP
8/26 Pcon-pT181 attenuator	1µL	10µL	11µL	Amp
8/26 Plac-pT181 antisense	1µL	10µL	11µL	CP
8/26 Pcon-pT181 antisense	1µL	10µL	11µL	Amp

Liquid culture

No name

Sample	medium
8/16 Plux	Plusgrow medium(+CP)

Genome PCR

Yoshdia and Nakagawa

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	SasA_fwd primer	SasA_rev primer	MilliQ	total
0.5	0.5	2.5	2.5	1.5	0.75	0.75	16	25

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	RpaA_fwd primer	RpaA_rev primer	MilliQ	total
0.5	0.5	2.5	2.5	1.5	0.75	0.75	16	25

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	RpaB_fwd primer	RpaB_rev primer	MilliQ	total
0.5	0.5	2.5	2.5	1.5	0.75	0.75	16	25

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	PkaiBC_fwd primer	PkaiBC_rev primer	MilliQ	total
0.5	0.5	2.5	2.5	1.5	0.75	0.75	16	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	50°C	68°C	--
2 min	10 sec	30 sec	38 sec	30 cycles

Ligation

No name

state	Vector		Inserter
experiment	8/27 DT (EcoRI & XbaI)	2.8	8/21 RBS-lysis3 (EcoRI & SpeI)
experiment	8/27 DT (EcoRI & XbaI)	2.8	8/27 pT181 attenuator (EcoRI & SpeI)

Samples were evaporeted used evaporator into about 3 µL.

sample	MilliQ	Ligation High	total
3	4	3.5	10.5

incubate overnight at 4 °C

Template:Kyoto/Notebook/Aug 27

From 2013.igem.org

Contents

Aug 27

Restriction Enzyme Digestion

Electrophoresis

Colony PCR

Liquid Culture

Restriction Enzyme Digestion

Electrophoresis

Gel Extraction

Transformation

Liquid culture

Genome PCR

Ligation