Template:Kyoto/Notebook/Sep 11

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Sep 11

PCR

Tatsui

pKaiBC(200p)	10*buffer KOD plus	dNTPs	MgSO4	primer mix(F&R)	KOD plus	MilliQ	total
0.5	2.5	2.5	1.5	1.5	0.5	16	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	60°C	68°C	30
2min	10sec	30sec	30sec

pKaiBC(400p)	10*buffer KOD plus	dNTPs	MgSO4	primer mix(F&R)	KOD plus	MilliQ	total
1	2.5	2.5	1.5	1.5	0.5	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	60°C	68°C	30
2min	10sec	30sec	30sec

pKaiBC(800p)	10*buffer KOD plus	dNTPs	MgSO4	primer mix(F&R)	KOD plus	MilliQ	total
2	2.5	2.5	1.5	1.5	0.5	14.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	60°C	68°C	30
2min	10sec	30sec	30sec

pKaiBC(1ng)	10*buffer KOD plus	dNTPs	MgSO4	primer mix(F&R)	KOD plus	MilliQ	total
2.5	2.5	2.5	1.5	1.5	0.5	14	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	60°C	68°C	30
2min	10sec	30sec	30sec

colony PCR

Tatsui

Sample	base pair
9/9Pcon-lacZα(p3B4k5)	712bp
9/9Pcon-spinach-DT(p3B4k5)	605bp

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	42s

Sample	base pair
Pbad\araC-RBS-luxI-DT

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

Sample	base pair
9/9Pcon+RBS-luxI-DT	1079bp
9/10Pcon+sptamer12_1R-DT	584bp
9/10DT+Pcon-pT181 attenuater 1	781bp
9/10Fusion1 attenuater(p3B1C3)-1	638bp

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
50sec	30sec	30sec	1min

Ligation

Okazaki

state	Vector		Inserter		Ligation High ver.2
experiment	9/8pSB4K5(EcoRl&Spel)21.8ng/µL	2.3 µL	9/8Pcon-Spinach-DT(EcoRl&Spel)10.5ng/µL	1.7 µL	4.0 µL
NC	9/8pSB4K5(EcoRl&Spel)21.8ng/µL	2.3 µL	9/8RBS-lacZα-DT(EcoRl&Spel)4.4ng/µL	6.7µL	9.0 µL
NC	9/8pSB4K5(EcoRl&Spel)21.8ng/µL	2.3 µL	9/8Pcon-RBS-lacZα-DT(EcoRl&Spel)5.6ng/µL	1.9 µL	4.2 µL
NC	9/6Plux 25ng/µL	2 µL	9/8 RBS-lysis1-DT(Xbal&Pstl)	6.6 µL	8.6 µL
NC	9/6Plux 25ng/µL	2 µL	9/8 RBS-lysis3-DT(Xbal&Pstl)	4.7 µL	6.7 µL
NC	9/10Pcon-pT181 attenuater(Spel&Pstl)	3.4µL	9/8RBS-lacZα-DT(EcoRl&Spel)	4.5 µL	7.9 µL
NC	9/6Pcon-RBS laxR-DT(EcoRl&Xbal) 26.7ng/µL	1.9 µL	Plax-RBS-GFP-DT	5.5 µL	7.4 µL
NC	9/9 Pcon-pT181 attenuater(SPpel&Pstl)	3.4 µL	9/10 aptamer12_1R-DT(Xbal&Pstl)	1.7 µL	5.1 µL

Transformation

Nakamoto Tatsui

Name

Sample

Competent Cells

Total

Plate

9/9 Fusion1 antisense(pSB2C3)

2µL

20µL

22µL

Amp

9/9 Fusion6 antisense(pSB2C3)

2µL

20µL

22µL

Amp

9/9 aptamer 12_P(pSB2C3)

2µL

20µL

22µL

Amp

9/9 aptamer 12_1&micro(pSB2C3)

2µL

20µL

22µL

Amp

9/9 Fusion2 attenuater(pSB2C3)

2µL

20µL

22µL

CP

9/11 Pcon-spinach-DT(pSB4K5)

2µL

20µL

22µL

Amp

9/11 RBS-lacZα-DT(pSB4K5)

2µL

20µL

22µL

Amp

9/11 Pcon-RBS-lacZα-DT(pSB4K5)

2µL

20µL

22µL

CP

Plux-RBS-lysis1-DT

2µL

20µL

22µL

Amp

Plux-RBS-lysis3-DT

2µL

20µL

22µL

Amp

2µL

20µL

22µL

CP

Plux-RBS-lysis1-DT

2µL

20µL

22µL

Amp

Plux-RBS-lysis3-DT

2µL

20µL

22µL

Amp

Plux

2µL

20µL

22µL

}

Restriction Enzyme Digestion

Nakamoto

	DNA	EcoRl	Xbal	Spel	Pstl	buffer	BSA	MilliQ	Total
pSB4K5(EcoRl+Spel)	9.4µL	1µL	0µL	1µL	0µL	3µL	3µL	12.6µL	30 µL
pSB4K5(EcoRl+Spel) negative	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL
RBS-lacZα-DT(EcoRl+Spel)	10µL	1µL	0µL	1µL	0µL	3µL	3µL	12µL	30µL
RBS-lacZα-DT(EcoRl+Spel) negative	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL
Ptet-pT181 antisense(Stel+Pstl)	10.8µL	0µL	0µL	1µL	1µL	3µL	3µL	12µL	30µL
Ptet-pT181 antisense(Stel+Pstl) negative	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL
Pcon-RBS-lacZα-DT(EcoRl+Spel)	7.1µL	1µL	0µL	1µL	0µL	3µL	3µL	14.9µL	30µL
Pcon-RBS-lacZα-DT(EcoRl+Spel) negative	0.4µL	0µL	0µL	0µL	0µL	1µL	1µL	7.6µL	10µL
RBS-lysis2-DT(Xbal+Pstl)	22µL	0µL	1µL	0µL	1µL	3µL	3µL	0µL	30µL
RBS-lysis2-DT(Xbal+Pstl) negative	1.4µL	0µL	0µL	0µL	0µL	1µL	1µL	6.6µL	10µL

Liquid Culture

Nakamoto

Sample	medium
8/16 Master4 Plax
8/25 Pλ-laxI-2

PCR

Hirano

8/29 Pλ-RBS-luxI-DT(76.5ng/µL)	10x buffer for KOD plus-ver.2	KOD-plus	dNTP	MgSO4	F primer(RBS-luxI-DT-cloning-fwd)	R primer(RBS-luxI-DT-cloning-rev)	MilliQ	total
0.3	2.5	0.5	2.5	1.5	0.75	0.75	16.2	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	65°C	68°C	--
5min	30sec	30sec	1min	30

8/31 Pbad/araC-RBS-RFP(1)(67.6ng/µL)	10x buffer for KOD plus-ver.2	KOD-plus	dNTP	MgSO4	F primer(Pbad/araC(pSBlc3)-cloning-fwd)	R primer(Pbad/araC(pSBlc3)-cloning-rev)	MilliQ	total
0.3	2.5	0.5	2.5	1.5	0.75	0.75	16.2	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	54°C	68°C	--
5min	30sec	30sec	3min 20sec	30

Liquid Culture

Hirano

Sample	medium
9/4 RBS-lysis1+DT 3	plusgrow(+CP) 3ml 37°C
9/4 RBS-lysis2+DT 1	plusgrow(+CP) 3ml 37°C
9/4 RBS-lysis3+DT 3	plusgrow(+CP) 3ml 37°C

Template:Kyoto/Notebook/Sep 11

From 2013.igem.org

Contents

Sep 11

PCR

colony PCR

Ligation

Transformation

Restriction Enzyme Digestion

Liquid Culture

PCR

Liquid Culture