Template:Kyoto/Notebook/Sep 11

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Sep 11

Electrophoresis

Lane	Sample
1	1kladder
2	Pbad/araC+RBS-luxI-DT-1
3	Pbad/araC+RBS-luxI-DT-1
4	Plac+RBS-lysis-DT
5	Pcon+RBS-luxI-DT
6	1kladder

File:Igku xxxxxx.xxx

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

Electrophoresis

Tatsui

Lane	Sample
1	100bp ladder
2	pKaiBC 800pg 60°C
3	pKaiBC 1ng 60°C
4	pKaiBC 800pg 61°C
5	pKaiBC 1ng 61°C
6	pKaiBC 800pg 62°C
7	pKaiBC 1ng 62°C
8	100bp ladder

File:Igku xxxxxx.xxx

colony PCR

Tatsui

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

Electrophoresis

Tatsui

Lane	Sample
1	100bp ladder
2	Pcon-lacZα(pSB4K5)3
3	Pcon-lacZα(pSB4K5)4
4	Pcon-lacZα(pSB4K5)5
5	Pcon-lacZα(pSB4K5)6
6	Pcon-spinach-DT(pSB4K5)3
7	Pcon-spinach-DT(pSB4K5)4
8	Pcon-spinach-DT(pSB4K5)5
9	Pcon-spinach-DT(pSB4K5)6
10	100bp ladder

File:Igku xxxxxx.xxx

Lane	Sample
1	1kbp ladder
2	Pbad/araC-RBS-luxI-DT3
3	Pbad/araC-RBS-luxI-DT4
4	Pbad/araC-RBS-luxI-DT5
5	Pbad/araC-RBS-luxI-DT6
6	1kbp ladder

File:Igku xxxxxx.xxx

Tatsui

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

Vector		Inserter		Ligation High ver.2
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
9/8pSB4K5(EcoRl&Spel)21.8ng/µL	2.3 µL	9/8RBS-lacZα-DT(EcoRl&Spel)4.4ng/µL	6.7µL	9.0 µL
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
9/6Plux 25ng/µL	2 µL	9/8 RBS-lysis1-DT(Xbal&Pstl)	6.6 µL	8.6 µL
9/6Plux 25ng/µL	2 µL	9/8 RBS-lysis3-DT(Xbal&Pstl)	4.7 µL	6.7 µL
9/10Pcon-pT181 attenuater(Spel&Pstl)	3.4µL	9/8RBS-lacZα-DT(EcoRl&Spel)	4.5 µL	7.9 µL
9/6Pcon-RBS laxR-DT(EcoRl&Xbal) 26.7ng/µL	1.9 µL	Plax-RBS-GFP-DT	5.5 µL	7.4 µL
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	CP
9/9 Fusion6 antisense(pSB2C3)	2µL	20µL	22µL	CP
9/9 aptamer 12_P(pSB2C3)	2µL	20µL	22µL	CP
9/9 aptamer 12_1&micro(pSB2C3)	2µL	20µL	22µL	CP
9/9 Fusion2 attenuater(pSB2C3)	2µL	20µL	22µL	CP
9/11 Pcon-spinach-DT(pSB4K5)	2µL	20µL	22µL	K
9/11 RBS-lacZα-DT(pSB4K5)	2µL	20µL	22µL	K
9/11 Pcon-RBS-lacZα-DT(pSB4K5)	2µL	20µL	22µL	K
Plux+RBS-lysis1-DT	2µL	20µL	22µL	CP
Plux+RBS-lysis3-DT	2µL	20µL	22µL	CP
Pcon-pT181attenuater+RBS-lacZα-DT	2µL	20µL	22µL	Amp
Pcon-iuxR+Plux-GFP	2µL	20µL	22µL	Amp
Pcon-pT181attenuater+apt12_1R-DT	2µL	20µL	22µL	Amp
Plac(BBa_R0011)	2µL	20µL	22µL	Amp

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

Electrophoresis

PCR

Electrophoresis

colony PCR

Electrophoresis

Ligation

Transformation

Restriction Enzyme Digestion

Liquid Culture

PCR

Liquid Culture