Template:Kyoto/Notebook/Sep 4

From 2013.igem.org

Sep 4

Ligation

No name

state	Vector		Inserter		Ligation High ver.2
experiment	9/3 DT(EcoRI&XbaI)	3.2µL	9/3 RBS-lysis1	2.4µL	2.7µL
experiment	9/3 DT(EcoRI&XbaI)	3.0µL	9/3 RBS-lysis2	4.7µL	4.0µL
experiment	9/3 DT(EcoRI&XbaI)	3.0µL	9/3 RBS-lysis3	8.8µL	4.0µL
experiment	9/3 DT(EcoRI&XbaI)	3.0µL	9/3 aptamer12_1R(EcoRI&SpeI)	1.9µL	2.45µL
experiment	9/2 pSB1C3(XbaI&PstI)	10.6µL	9/3 pT181attenuator (XbaI& PstI)	3.2µL	4.0µL
experiment	9/2 pSB1C3(XbaI&PstI)	9.6µL	9/3 pT181antisense (XbaI& PstI)	2.1µL	4.0µL
experiment	9/2 pSB1C3(EcoRI&SpeI)	16.7µL	9/3 aptamer12_1R(EcoRI&SpeI)	2.0µL	4.0µL

incubate 16 °C 1 hour

Transformation

Tatsui

Name	Sample	Competent Cells	Plate
9/4 RBS-lysis1-DT	2µL	20µL	Amp
9/4 RBS-lysis2-DT	2µL	20µL	Amp
9/4 RBS-lysis3-DT	2µL	20µL	Amp
9/4 aptamer12_1R-DT	2µL	20µL	Amp
9/4 pT181attenuator –pSB1C3	2µL	20µL	Amp
9/4 pT181antisense-pSB1C3	2µL	20µL	Amp
9/4 aptamer12_1R –pSB1C3	2µL	20µL	Amp
9/4 pSB4K5(2013plate5 56)	2µL	20µL	Amp
9/3 Mutagenesis product 1(KaiABC)	2µL	20µL	Amp
9/3 Mutagenesis product 2(NC)	2µL	20µL	Amp

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
DT	193.1	1.93	1.81
Spinach-DT	373.3	1.92	1.69
RBS-lysis1	332.0	1.95	1.62

Master Plate

Hirano

Number	Use LB plate (+CP)
1	8/10 Plac(CP)-2
2	8/10 Plac(CP)-2
3	8/10 Plac(CP)-2

Number	Use LB plate (+Amp)
1	8/9 J23100(Amp)-1
2	8/9 J23100(Amp)-1
3	8/9 J23100(Amp)-1

Liquid Culture

Hirano

Sample	medium
8/10 Plac(CP)-2	Plusgrow medium(+CP)
8/9 J23100(Amp)-1	Plusgrow medium(+Amp)

PCR

Hirano

8/29 Pλ-RBS-luxI-DT	KOD plus	10x buffer	dNTP	MgSO4	F primer (RBS-luxI-DT-cloning-fwd)	R primer(RBS-luxI-DT-cloning-rev)	MilliQ	total
0.3µL	0.5µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	16.2µL	25µL

8/31 Pbad/araC-RBS-RFP(1)	KOD plus	10x buffer	dNTP	MgSO4	F primer (Pbad/araC-pSB1C3-cloning-fond)	R primer(Pbad/araC-cloning-rex)	MilliQ	total
0.3µL	0.5µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	16.2µL	25µL

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	65°C	68°C	--
5min	10s	30s	3min10s	30cycles

Restriction Enzyme Digestion

No name

	8/21 Pconst	SpeI	PstI	Buffer	BSA	MilliQ	total
2 cuts	10.5µL	1.0µL	1.0µL	3.0µL	0.3µL	14.2µL	30 µL
NC	0.5µL	0µL	0µ	1.0µL	0.1µL	8.4µL	10 µL

	8/29 Plac	SpeI	PstI	Buffer	BSA	MilliQ	total
2 cuts	13.0µL	1.0µL	1.0µL	3.0µL	0.3µL	11.7µL	30 µL
NC	0.7µL	0µL	0µ	1.0µL	0.1µL	8.2µL	10 µL

	8/20 Ptet	SpeI	PstI	Buffer	BSA	MilliQ	total
2 cuts	9.0µL	1.0µL	1.0µL	3.0µL	0.3µL	15.7µL	30 µL
NC	0.5µL	0µL	0µ	1.0µL	0.1µL	8.4µL	10 µL

	8/20 RBS-tetR-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	8.5µL	1.0µL	1.0µL	3.0µL	0.3µL	16.2µL	30 µL
NC	0.5µL	0µL	0µ	1.0µL	0.1µL	8.4µL	10 µL

	spinach -DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	5.4µL	1.0µL	1.0µL	3.0µL	3.0µL	16.6µL	30 µL
NC	0.3µL	0µL	0µ	1.0µL	1.0µL	7.7µL	10 µL

	8/17 RBS-lacZα-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	11µL	1.0µL	1.0µL	3.0µL	3.0µL	11µL	30 µL
NC	0.5µL	0µL	0µ	1.0µL	1.0µL	7.5µL	10 µL

Transformation

No name

Name	Sample	Competent Cells	Total	Plate
Ptrc KaiC	1µL	10µL	11µL	Amp
Ptrc KaiB	1µL	10µL	11µL	Amp
pSΩ	1µL	10µL	11µL	Amp

Electrophoresis

No name
1

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Pconst	SpeI	PstI
3	Pconst NC	--	--
4	Plac	SpeI	PstI
5	Plac NC	--	--
6	100bp ladder	--	--

2

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Ptet	SpeI	PstI
3	Ptet NC	--	--
4	RBS-tetR-DT	XbaI	PstI
5	RBS-tetR-DT NC	--	--
6	100bp ladder	--	--

3

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Spinach-DT	XbaI	PstI
3	Spinach-DT NC	--	--
4	RBS-lacZα-DT	XbaI	PstI
5	RBS-lacα-DT NC	--	--
6	100bp ladder	--	--

Gel Extraction

No name

1

Lane	DNA	Enzyme
1	100bp ladder	--
2	Pcon	SpeI&PstI
3	Pcon	SpeI&PstI
4	--	--
5	Plac	SpeI&PstI
6	Plac	SpeI&PstI

2

Lane	DNA	Enzyme
1	100bp ladder	--
2	Ptet	SpeI&PstI
3	Ptet	SpeI&PstI
4	--	--
5	RBS-tetR-DT	XbaI&PstI
6	RBS-tetR-DT	XbaI&PstI

Name	quantity	concentration[µg/mL]	260/280	260/230
Pcon	--	13.2	1.99	-1.64
Plac	--	15.3	2.07	-0.39
Ptet	--	30.1	1.97	-0.97
RBS-tetR-DT	--	3.0	2.44	0.88

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder	--
2	spinach-DT	XbaI&PstI
3	spinach-DT	XbaI&PstI
4	--	--
5	RBS-lacZα-DT	XbaI&PstI
6	RBS-lacZα-DT	XbaI&PstI

Name	quantity	concentration[µg/mL]	260/280	260/230
spinach-DT	--	2.1	2.10	-0.05
RBS-lacZα-DT	--	3.3	2.59	-0.07

Master Plate

No name

Number	Use LB plate (+Amp)
4	8/9 BBa_J23100-3
5	8/9 BBa_J23100-4
6	8/9 BBa_J23100-5

Number	Use LB plate
1	JM109-1
2	JM109-2
3	JM109-3
4	JM109-4

Liquid Culture

Hirano

Sample	medium
8/9 BBa_J23100-3	Plusgrow medium(+Amp)
JM109-1(Master plate)	LB medium

Colony PCR

Hirano

Sample	base pair
8/9 BBa_J23100 3	--
8/9 BBa_J23100 4	--
8/9 BBa_J23100 5	--

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

Template:Kyoto/Notebook/Sep 4

From 2013.igem.org

Contents

Sep 4

Ligation

Transformation

Miniprep

Master Plate

Liquid Culture

PCR

Restriction Enzyme Digestion

Transformation

Electrophoresis

Gel Extraction

Gel Extraction

Master Plate

Liquid Culture

Colony PCR