Template:Kyoto/Notebook/Sep 10

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Sep 10

Ligation

Nakamoto

state	Vector		Inserter		Ligation High ver.2
experiment	9/6 Pcon-RBS-luxR-DT	1.9µL	9/6Plux-RBS-GFP-DT	22 µL	µL
experiment	9/6 Plux	2µL	9/8 RBS-lysis1-DT	6.6µL	xx µL
experiment	9/6 Plux	2µL	9/8 RBS-lysis2-DT	6.3µL	xx µL
experiment	9/8 Ptet-PT181 antisense	1.6µL	9/8 spinach-DT	4.4µL	xx µL
experiment	9/8 Pcon-PT181 attenuator	3.4µL	9/8 apz 12-1R-PT	2.1µL	xx µL
experiment	9/8 Pcon	3.4µL	9/8 apz 12-1R-PT	2.3µL	xx µL
experiment	9/8 DT	3.0µL	9/8 Pcon-PT181 attenuator	7.9µL	xx µL
experiment	9/10 pSB1C3	1.8µL	9/8 F3m2 attenuator	4.6µL	xx µL
experiment	9/10 pSB1C3	1.8µL	9/8 F1 attenuator	3.4µL	xx µL

Miniprep

Nakamoto

DNA	concentration[µg/mL]	260/280	260/230
spinach-DT	202.4	1.97	1.93
Plux-RBS-GFP-DT	164.2	2.80	2.06
Pcon-DT attenuator	205.4	1.81	2.39
RSBJC3-2	240.8	1.73	1.43
Plaz-1	240.8	1.73	1.43

Restriction Enzyme Digestion

Nakamoto

	9/10　Plux-RBS-GFP-DT	EcoR1	Spel	Buffer	BSA	MilliQ	total
2cuts	12.1µL	1µL	1µL	3µL	3µL	9.9µL	30µL
NC	0.6µL	0µL	0µL	1µL	1µL	7.4µL	10µL

	9/10　Spinach-DT	Xbal	Pstl	Buffer	BSA	MilliQ	total
2cuts	9.9µL	1µL	1µL	3µL	3µL	12.1µL	30µL
NC	0.5µL	0µL	0µL	1µL	1µL	7.5µL	10µL

	9/6 apt12-1R -DT	Xbal	Pstl	Buffer	BSA	MilliQ	total
2cuts	8.0µL	1µL	1µL	3µL	3µL	14.0µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/10　Pcon-pt81 attenuator	EcoR1	Spel	Buffer	BSA	MilliQ	total
2cuts	9.8µL	1µL	1µL	3µL	3µL	12.2µL	30µL
NC	0.5µL	0µL	0µL	1µL	1µL	7.5µL	10µL

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Plux-RBS-GEP-DT	EcoR1	Spel
3	Plux-RBS-GEP-DT	--	--
4	Spinach-DT	Xbal	Pstl
5	Spinach-DT	--	--
6	aptamer 121R-DT	Xbal	Pstl
7	aptamer 121R-DT	--	--
8	Pcon-pt181 attenuator	EcoR1	Spel
9	Pcon-pt181 attenuator	--	--
10	100bp ladder	--	--

Colony PCR

Tatsui

Sample	base pair
9/9 Pcon-pT181 antisense	--
9/8 J223100(entA)	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min10sec	30

Electrophoresis

Tatsui

Lane	Sample
1	100bp ladder
2	Pcon Pt181 antisense1+spinach-DT
3	Pcon Pt181 antisense2+spinach-DT
4	Pcon Pt181 antisense3+spinach-DT
5	Pcon Pt181 antisense4+spinach-DT
6	Pcon Pt181 antisense5+spinach-DT
7	Pcon Pt181 antisense6+spinach-DT
8	Pcon Pt181 antisense7+spinach-DT
9	Pcon Pt181 antisense8+spinach-DT
10	Pcon Pt181 antisense9+spinach-DT
11	Pcon Pt181 antisense10+spinach-DT
12	Pcon Pt181 antisense11+spinach-DT
13	Pcon Pt181 antisense12+spinach-DT
14	100bp ladder

Lane	Sample
1	1kbp ladder
2	Pcon entA1
3	Pcon entA2
4	Pcon entA3
5	1kbp ladder

Gel Extraction

Tatsui

Lane	DNA	Enzyme
1	1kbp ladder	--
2	KaiA(200pg)	--
3	KaiA(200pg)	--
4	--	--
5	KaiA(400pg)	--
6	KaiA(400pg)	--
7	--	--
8	KaiA(800pg	--
9	KaiA(800pg)	--
10	1kbp ladder	--

Lane	DNA	Enzyme
1	1kbp ladder	--
2	PKaiBC(200pg)	--
3	PKaiBC(200pg)	--
4	--	--
5	PKaiBC(400pg)	--
6	PKaiBC(400pg)	--
7	--	--
8	PKaiBC(800pg	--
9	PKaiBC(800pg)	--
10	1kbp ladder	--

Liquid Culture

Hirano

Sample	medium
9/4 J23100①(JM109)	LB(Amp)
9/8 J23100① (entA)	LB(Amp)

Gel Extraction

Tatsui

Lane	DNA	Enzyme
1	1kbp ladder	--
2	KaiC	--
3	KaiC	--
4	KaiC	--
5	--	--
6	1kbp ladder	--
7	1kbp ladder	--
8	KaiC	--
9	KaiC	--
10	KaiC	--
11	--	--
12	1kbp ladder	--

Liquid Culture

Tatsui

Sample	medium
9/10 Pcon-pt181 antisense-Spinach-DT	Plusgrow

Colony PCR

No name

Sample	base pair
9/9 Phad larac-1 RBS-lux1-DT	2331

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

Sample	base pair
9/9 Pcon-lacZα (psB4KS)	712
9/9 Pcon-Spinach-DT(psB4KS)	605

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

Sample	base pair
9/9 Plac+RBS-lysis3}-DT	1273
9/9 Pcon+RBS-lux1-DT	1079

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min12s	30

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	9/9 Pcou-RBS-lacZα-DT(p3B4KS9)-1	--	--
3	9/9 Pcou-RBS-lacZα-DT(p3B4KS9)-2	--	--
4	9/9 Pcou-Spinach-DT(pB4KS)-1	--	--
5	9/9 Pcou-Spinach-DT(pB4KS)-2	--	--
6	100bp ladder	--	--

Transformation

Nakamoto

Name	Plate
F1 attenuator +p3B1C3	CP
F3n2 attenuator +p3B1C3	CP
F1 antisense +p3B1C3	CP
F6 antisense+p3B1C3	CP
apt12-P +p3B1C3	CP
apt12-1M +p3B1C3	CP
Pcon-luxR+Plux-GFP	Amp
Plux+RBS-lysis1-DT	CP
Plux+RBS-lysis2-DT	CP
Ptet-pT181antisense+Spinach-DT	CP
Pcon-pT181 attenuator+apt121R-DT	Amp
Pcon-1 apt12-1R-DT	Amp
DT+Pcon-pT181 attenuator	CP

Electrophoresis

Okazaki

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Spinach-DT	Xbal	Ptsl
3	Spinach-DT	Xbal	Ptsl
4	Spinach-DT	Xbal	Ptsl
5	Spinach-DT	Xbal	Ptsl
6	100bp ladder	--	--

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Pcon-pT181 attenuator	EcoR1	Spel
3	Pcon-pT181 attenuator	EcoR1	Spel
4	Pcon-pT181 attenuator	EcoR1	Spel
5	Pcon-pT181 attenuator	EcoR1	Spel
6	100bp ladder	--	--

Lane	Sample	Enzyme1	Enzyme2
1	Plux-RBS-GFP-DT	EcoR1	Spel
2	Plux-RBS-GFP-DT	EcoR1	Spel
3	Plux-RBS-GFP-DT	EcoR1	Spel
4	Plux-RBS-GFP-DT	EcoR1	Spel
5	100bp ladder	--	--
6	apt12-1R-DT	Xbal	Pstl
7	apt12-1R-DT	Xbal	Pstl
8	apt12-1R-DT	Xbal	Pstl
9	apt12-1R-DT	Xbal	Pstl

Template:Kyoto/Notebook/Sep 10

From 2013.igem.org

Contents

Sep 10

Ligation

Miniprep

Restriction Enzyme Digestion

Electrophoresis

Colony PCR

Electrophoresis

Gel Extraction

Liquid Culture

Gel Extraction

Liquid Culture

Colony PCR

Electrophoresis

Transformation

Electrophoresis