Template:Kyoto/Notebook/Sep 14

From 2013.igem.org

Sep 14

Restriction Enzyme Digestion

Hirano

	9/14 pSB4K5S	EcoRI	XbaI	SpeI	PstI	BufferB	BufferD	BSA	MilliQ	total
2cuts	10µL	1µL	0µL	1µL	0µL	3µL	0µL	3µL	12µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	0µL	1µL	7.5µL	10µL

	9/13 Plac	XbaI	PstI	BufferH	BSA	MilliQ	total
1 cut	9µL	0µL	1µL	3µL	3µL	14µL	30µL
NC	1µL	0µL	0µL	1µL	1µL	7µL	10µL

	9/14 PKaiBC	EcoRI	XbaI	SpeI	PstI	BufferB	BSA	MilliQ	total
2cuts	11µL	1µL	0µL	1µL	0µL	3µL	3µL	11µL	30µL
NC	4µL	0µL	0µL	0µL	0µL	1µL	1µL	4µL	10µL

	9/14 RpaB	EcoRI	XbaI	SpeI	PstI	BufferD	BSA	MilliQ	total
2cuts	13µL	0µL	1µL	0µL	1µL	3µL	3µL	9µL	30µL
NC	2µL	0µL	0µL	0µL	0µL	1µL	1µL	5µL	10µL

	8/17 RBS-GFP-DT	EcoRI	XbaI	SpeI	PstI	BufferD	BSA	MilliQ	total
1cut	9µL	0µL	1µL	0µL	0µL	3µL	3µL	14µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL

incubated 37°C 1hour

Liquid Culture

Hirano

Sample	medium
9/11 Plac(BBa-R0011)-(3)	Plusgrow medium(+Amp)

Electrophoresis

Hirano

Lane	Sample	Enzyme1	Enzyme2
1	1kb ladder	--	--
2	pSB4K5(1)	EcoRI	SpeI
3	pSB4K5(1)	--	--
4	Plac(1A2)	PstI	--
5	Plac(1A2)	--	--
6	PKaiBC	EcoRI	SpeI
7	PKaiBC	--	--
8	RpaB(1)	XbaI	PstI
9	RpaB(1)	--	--
10	RBS-GFP-DT	XbaI	--
11	RBS-GFP-DT	--	--
12	1kb ladder	--	--

Lane	Sample	Enzyme
1	1kb ladder	--
2	Plac(pSB1A2)	PstI
3	Plac(pSB1A2)	--

Sequence PCR

No name

genome DNA	Big Dye	5x buffer	temp(400mg)	primer	milliQ
Pcon-RBS-luxR-DT	0.5	1.75	0.9	0.5	6.85
Pcon-RBS-luxR-DT	0.5	1.75	0.4	0.5	7.35
Pcon-RBS-tetR-DT	0.5	1.75	3	0.5	5.25
Pcon-RBS-tetR-DT	0.5	1.75	3	0.5	5.25
Plux	0.5	1.75	2.4	0.5	5.35
Plux-RBS-GFP-DT	0.5	1.75	1.9	0.5	5.85
Plux-RBS-GFP-DT	0.5	0.5	1.9	0.5	5.85
Pcon-RBS-GFP-DT	0.5	0.5	1.2	0.5	6.55
Pcon-RBS-GFP-DT	0.5	0.5	1.2	0.5	6.55
RBS-lysis3-DT	0.5	0.5	1.0	0.5	6.75
Pcon-antisense-Spinach-DT	0.5	0.5	1.4	0.5	6.35
Pcon-RBS lacZα-DT	0.5	0.5	1.4	0.5	6.35
Ptet-RBS lacZα-DT	0.5	0.5	0.59	0.5	1.85

PreDenature	Denature	Annealing	Extension	cycle
96°C	96°C	50°C	60°C	--
2min	10sec	5sec	4min	30cycle

Gel Extraction

Tatsui

Lane	DNA	Enzyme
1	1kb ladder	--
2	pSB4K5	EcoRI&SpeI
3	pSB4K5	EcoRI&SpeI
4	pSB4K5	EcoRI&SpeI

Restriction Enzyme Digestion

No name

	9/14 Plac(1A2)	EcoRI	XbaI	SpeI	PstI	BufferB	BSA	MilliQ	total
2cuts	23µL	0µL	0µL	0.3µL	0µL	3µL	3µL	0µL	30µL

	9/14 RBS-GFP-DT	EcoRI	XbaI	SpeI	PstI	BufferH	BSA	MilliQ	total
2 cuts	23µL	1µL	0µL	0µL	0µL	3µL	3µL	0µL	30µL

Ligation

Nakamoto and Hirano

state	Vector		Inserter		Ligation High ver.2
experiment	9/13 Pcon-PT181attenuator(SpeI&PstI)	1.7	9/13 RBS-lacZα-DT (XbaI & PstI)	12.5	5
experiment	9/13 pSB2C3 (XbaI & PstI)	1.9	9/14 RpaB (XbaI & PstI)	16.3	10.5
experiment	9/13 pSB2C3 (XbaI & PstI)	4.7	9/14 PkaiBC (EcoRI & SpeI)	6.7	5.7
experiment	9/14 RBS-GFP-DT (PstI&XbaI)	0.7	9/14 PkaiBC (EcoRI & SpeI)	2.6	1.6
experiment	9/14 Plac (SpeI&PstI)	2.4	9/14 RBS-lysis1-DT (XbaI & PstI)	4.9	3.6
experiment	9/14 Plac (SpeI&PstI)	2.4	9/14 RBS-lysis2-DT (XbaI & PstI)	7.0	4.7
experiment	9/14 Plac (SpeI&PstI)	2.4	9/14 RBS-lysis3-DT (XbaI & PstI)	9.2	5.8

Samples were evaporeted used evaporator into about 7 µL

Colony PCR

No name

Sample	base pair
9/13 Plux＋RBS-lysis1-DT (1)~(3)	--
9/13 Plux＋RBS-lysis2-DT (1)~(3)	-
9/13 Plux＋RBS-lysis3-DT (1)~(2)	-

Sample	base pair
9/13 Pcon-RBS-luxR-DT＋Plux-RBS-GFP-DT (1)~(4)	--
9/13 Pcon-PT181attenuator＋aptamer121R-DT (1)~(2)	-
9/13 Plac＋PT181attenuator (1)	-

Sample	base pair
Dλ-lux(1) (1)~(2)	--
NC	--

Electrophoresis

No name

Lane	Sample
1	1kbp ladder
2	9/13 Plux+RBS-lysis1-DT1
3	9/13 Plux+RBS-lysis1-DT2
4	9/13 Plux+RBS-lysis1-DT3
5	9/13 Plux+RBS-lysis2-DT1
6	9/13 Plux+RBS-lysis2-DT2
7	9/13 Plux+RBS-lysis2-DT3
8	9/13 Plux+RBS-lysis3-DT1
9	9/13 Plux+RBS-lysis3-DT2
10	9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT1
11	9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT2
12	9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT3
13	9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT4
14	9/13 Ptet-pT181 antisense+Spinach-DT1
15	9/13 Pcon-pT181 attenuator+aptamer12-1R-PT1
16	9/13 Pcon-pT181 attenuator+aptamer12-1R-PT1
17	9/13 Plac+pT181 attenuator

Lane	Sample
1	1kbp ladder
2	Pλ-lux(1)1
3	Pλ-lux(1)1
4	NC

Liquid Culture

No name

Sample	medium
9/13 Pλ-luxI(1)-1	Plusgrow medium(+Amp)

Transformation

Nakamoto

Name	Sample	Competent Cells	Total	Plate
9/13 RBS-lysisZα-DT(XbaI&PstI)＋9/13 Pcon-PT181 attenuator(SpeI&PstI)	2µL	20µL	22µL	Amp
9/13 SB2C3＋9/14 PzαB	2µL	20µL	22µL	CP
9/13 pSB2C3＋9/14 PkaiBC	2µL	20µL	22µL	CP
9/13 RBS-GFP-DT＋9/14 PkaiBC	2µL	20µL	22µL	CP
Plac＋RBS-lysis1-DT	2µL	20µL	22µL	Amp
Plac＋RBS-lysis2-DT	2µL	20µL	22µL	Amp
Plac＋RBS-lysis2-DT	2µL	20µL	22µL	Amp

Sequence PCR

Tatsui

genome DNA	temp	Big Dye	5x buffer	primer	MilliQ
9/6 PT181 attenuator(VR)	3.9	0.5	1.75	0.5	3.85
8/17 RBS lacZα-DT	2.1	0.5	1.75	0.5	5.65
9/6 PT181 attenuator(VR)	3.9	0.5	1.75	0.5	3.85

PreDenature	Denature	Annealing	Extension	cycle
96°C	50°C	60°C	4°C	--
10sec	5sec	4min	1min	30cycle

Template:Kyoto/Notebook/Sep 14

From 2013.igem.org

Contents

Sep 14

Restriction Enzyme Digestion

Liquid Culture

Electrophoresis

Sequence PCR

Gel Extraction

Restriction Enzyme Digestion

Ligation

Colony PCR

Electrophoresis

Liquid Culture

Transformation

Sequence PCR