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	2.3 µL	Pcon-Spinach-DT(EcoRl&Spel)10.5ng/µL	1.7 µL	4.0 µL
NC	9/8pSB4K5(EcoRl&Spel)21.8ng/µL	xx µL	name	xx µL	xx µL
NC	name	xx µL	name	xx µL	xx µL
NC	name	xx µL	name	xx µL	xx µL
NC	name	xx µL	name	xx µL	xx µL
NC	name	xx µL	name	xx µL	xx µL
NC	name	xx µL	name	xx µL	xx µL
NC	name	xx µL	name	xx µL	xx µL

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

Restriction Enzyme Digestion

Liquid Culture

PCR

Liquid Culture