Template:Kyoto/Notebook/Sep 3

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Sep 3

Electrophoresis

Kozima

Lane	Sample
1	100bp ladder
2	pT181 attenuator(2)-DT2(738)
3	Ptet-RBS-lacZα-DT-2(756)
4	Ptet-RBS-lacZα-DT-3
5	Ptet-RBS-lacZα-DT-4
6	Ptet-RBS-lacZα-DT-5
7	Ptet-RBS-lacZα-DT-6
8	RBS-lysis2-DT-2(985)
9	RBS-lysis2-DT-3
10	100bp ladder

Miniprep

Tatsui

DNA	concentration[µg/mL]	260/280	260/230
9/2 14:00 pT181 attenuator	418.4	1.82	1.80
9/2 14:00 pT181 antisense	362.2	1.92	1.96
9/2 14:00 Spinach	438.3	1.86	1.99
9/2 14:00 tetR-apt 12_1R	567.5	1.90	1.99
9/2 21:00 pT181 attenuator	320.9	1.90	2.15
9/2 21:00 Spinach	457.0	1.88	2.24

Liquid Culture

Kozima

Sample	medium
Spinach-DT(9/2 master plate)	plusgrow(CP)4ml

Restriction Enzyme Digestion

Kozima

	DNA	EcoR1	Spe1	XbaI	Pst1	10xBuffer B(EcoR1&Spe1)	10xBuffer D(Xba1&Pst1)	100xBSA	MilliQ	total
9/3 pT181attenuator 14:00 418.4ng/µL	4.8µL	1.0µL	1.0µL	0µL	0µL	3µL	0µL	0.3µL	19.9µL	30µL
9/3 pT181attenuator 14:00 418.4ng/µL	0.2µL	0µL	0µL	0µL	0µL	1µL	0µL	0.1µL	8.7µL	10µL
9/3 pT181attenuator 14:00 418.4ng/µL	4.8µL	0µL	0µL	1.0µL	1.0µL	0µL	3µL	0.3µL	19.9µL	30µL
9/3 pT181attenuator 14:00 418.4ng/µL	0.2µL	0µL	0µL	0µL	0µL	0µL	1µL	0.1µL	8.7µL	10µL

	DNA	EcoR1	Spe1	Xba1	Pst1	10xBuffer B(EcoR1&Spe1)	10xBuffer D(Xba1&Pst1)	100xBSA	MilliQ	total
9/3 pT181antisense 14:00 362.2ng/µL	5.5µL	1.0µL	1.0µL	0µL	0µL	3µL	0µL	0.3µL	19.2µL	30µL
9/3 pT181attenuator 14:00 418.4ng/µL	0.3µL	0µL	0µL	0µL	0µL	1µL	0µL	0.1µL	8.6µL	10µL
9/3 pT181attenuator 14:00 418.4ng/µL	5.5µL	0µL	0µL	1.0µL	1.0µL	0µL	3µL	0.3µL	19.2µL	30µL
9/3 pT181attenuator 14:00 418.4ng/µL	0.3µL	0µL	0µL	0µL	0µL	0µL	1µL	0.1µL	8.6µL	10µL

	DNA	EcoR1	Spe1	10xBuffer B	100xBSA	MilliQ	total
9/3 apt 12_1R 14:00 507.5ng/µL	3.9µL	1.0µL	1.0µL	3µL	0.3µL	20.8µL	30µL
9/3 apt 12_1R 14:00 507.5ng/µL	0.2µL	0µL	0µL	1µL	0.1µL	8.7µL	10µL

	DNA	EcoRI	XbaI	10xBuffer D	100xBSA	MilliQ	total
8/29 DT 166.2ng/µL	12µL	1.0µL	1.0µL	3µL	0.3µL	12.7µL	30µL
8/29 DT 166.2ng/µL	0.6µL	0µL	0µL	1µL	0.1µL	8.3µL	10µL

	DNA	EcoRI	SpeI	10xBuffer B	100xBSA	MilliQ	total
8/22 RBS-lysis1(2) 96ng/µL	21µL	1.0µL	1.0µL	3µL	0.3µL	3.7µL	30µL
8/22 RBS-lysis1(2) 96ng/µL	1.0µL	0µL	0µL	1µL	0.1µL	7.9µL	10µL

	DNA	EcoRI	SpeI	10xBuffer B	100xBSA	MilliQ	total
8/30 RBS-lysis2 220ng/µL	9.1µL	1.0µL	1.0µL	3µL	0.3µL	15.6µL	30µL
8/30 RBS-lysis2 220ng/µL	0.5µL	0µL	0µL	1µL	0.1µL	8.4µL	10µL

	DNA	EcoRI	SpeI	10xBuffer B	100xBSA	MilliQ	total
8/20 RBS-lysis3(1) 282ng/µL	7.1µL	1.0µL	1.0µL	3µL	0.3µL	17.6µL	30µL
8/20 RBS-lysis3(1) 282ng/µL	0.4µL	0µL	0µL	1µL	0.1µL	8.5µL	10µL

PCR

Hirano

Plac(1) plasmid 153.7ng/µL	10x buffer KOD plus-ver.2	2mM dNTPs	25mM MgSO4	primer (KaiA_BsaI_Mut_fwd)	primer (KaiA_BsaI_Mut_rer)	KOD plus-ver.2	MilliQ	total
0.13	2.5	2.5	1.5	0.75	0.75	0.5	16.37	25

KaiB (plasmid) 20.2ng/µL	10x buffer KOD plus-ver.2	2mM dNTPs	25mM MgSO4	primer (GG2_RBS_KaiB_fwd)	primer (KaiB_GG3_rer)	KOD plus-ver.2	MilliQ	total
1	2.5	2.5	1.5	0.75	0.75	0.5	15.5	25

KaiC (plasmid) 4.4ng/µL	10x buffer KOD plus-ver.2	2mM dNTPs	25mM MgSO4	primer	primer	KOD plus-ver.2	MilliQ	total
1	2.5	2.5	1.5	0.75	0.75	0.5	15.5	25

DT-1 (plasmid) 153.7ng/µL	10x buffer KOD plus-ver.2	2mM dNTPs	25mM MgSO4	primer	primer	KOD plus-ver.2	MilliQ	total
0.1	2.5	2.5	1.5	0.75	0.75	0.5	16.4	25

PKaiBC(PCR product) 16.3ng/µL	10x buffer KOD plus-ver.2	2mM dNTPs	25mM MgSO4	primer	primer	KOD plus-ver.2	MilliQ	total
0.7	2.5	2.5	1.5	0.75	0.75	0.5	15.8	25

SasA(PCR product) 21.6ng/µL	10x buffer KOD plus-ver.2	2mM dNTPs	25mM MgSO4	primer	primer	KOD plus-ver.2	MilliQ	total
0.5	2.5	2.5	1.5	0.75	0.75	0.5	16	25

RpaA(PCR product) 15.4ng/µL	10x buffer KOD plus-ver.2	2mM dNTPs	25mM MgSO4	primer	primer	KOD plus-ver.2	MilliQ	total
0.6	2.5	2.5	1.5	0.75	0.75	0.5	15.9	25

RpaB(PCR product) 26.7ng/µL	10x buffer KOD plus-ver.2	2mM dNTPs	25mM MgSO4	primer	primer	KOD plus-ver.2	MilliQ	total
0.4	2.5	2.5	1.5	0.75	0.75	0.5	16.1	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C		68°C	--
5min	10sec	30sec		30

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C		68°C	--
5min	10sec	30sec		30

Mutatation PCR

No name

KaiABC(12.6pg/µL)	Primer(Kai_BsaIMut_fwd)	Primer(Kai_BsaIMut_rer)	Prime STAR Max Premix	MilliQ	total
1	1	1	25	22	50

	Primer(Kai_BsaIMut_fwd)	Primer(Kai_BsaIMut_rer)	Prime STAR Max Premix	MilliQ	total
	1	1	25	23	50

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	65°C	72°C	--
5min	10sec	15sec	40sec	30

Colony PCR

Sample	base pair
9/1 pT181antisense(pSB1C3)	400bp
9/1 pT181antisense-DT	547bp

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

Electrophoresis

Kozima

Lane	Sample
1	100bp ladder
2	9/1 pT181antisense(pSB1C3)
3	9/1 pT181antisense

Electrophoresis

Lane	Sample
1	100bp ladder
2	attenuator EcoR1&Spe1
3	attenuator negative EcoR1&Spe1
4	attenuator Xba1&Pst1
5	attenuator negative Xba1&Pst1
6	antisense EcoR1&Spe1
7	antisense negative EcoR1&Spe1
8	100bp ladder

Lane	Sample
1	100bp ladder
2	antisense Xba1&Pst1
3	antisense negative Xba1&Pst1
4	apt EcoR1&Spe1
5	apt negative EcoR1&Spe1
6	DT EcoR1&Xba1
7	DT negative EcoR1&Xba1
8	100bp ladder

Lane	Sample
1	100bp ladder
2	RBS-lysis1 EcoR1&Spe1
3	RBS-lysis1 negative
4	RBS-lysis2 EcoR1&Spe1
5	RBS-lysis2 negative
6	RBS-lysis3 EcoR1&Spe1
7	RBS-lysis3 negative
8	100bp ladder

Gel Extraction

Lane	DNA	Enzyme
1	100bp ladder
3	pT181 attenuator	EcoRI&SpeI
4	pT181 attenuator	EcoRI&SpeI
6	pT181 attenuator	XbaI&PstI
7	pT181 attenuator	XbaI&PstI
9	pT181 antisense	EcoRI&SpeI
10	pT181 antisense	EcoRI&SpeI

Lane	DNA	Enzyme
1	100bp ladder
2	pT181 antisense	XbaI&PstI
3	pT181 antisense	XbaI&PstI
5	apt12_1R	EcoRI&SpeI
6	apt12_1R	EcoRI&SpeI

Lane	DNA	Enzyme
1	100bp ladder
2	DT	EcoRI&XbaI
3	DT	EcoRI&XbaI
5	EcoRI&SpeI
6	RBS-lysis1	EcoRI&SpeI

Lane	DNA	Enzyme
1	100bp ladder
2	RBS-lysis2	EcoRI&SpeI
3	RBS-lysis2	EcoRI&SpeI
5	RBS-lysis3	EcoRI&SpeI
6	RBS-lysis3	EcoRI&SpeI

Name	concentration[µg/mL]	260/280	260/230
pT181 attenuator(EcoRI&SpeI)	10.0	1.86	0.39
pT181 attenuator(XbaI&PstI)	11.7	1.65	0.36
pT181 antisense(EcoRI&SpeI)	4.2	1.95	0.25
pT181 antisense(XbaI&PstI)	6.6	1.78	0.33
tRNA 12_1R apt(EcoRI&SpeI)	5.7	1.73	0.29
DT(EcoRI&XbaI)	16.6	1.99	0.76
RBS-lysis1(EcoRI&SpeI)	8.8	1.65	0.32
RBS-lysis2(EcoRI&SpeI)	13.4	1.61	0.48
RBS-lysis3(EcoRI&SpeI)	10.2	2.19	0.51

Liquid Culture

No name

Sample	medium
8/16master plate7(DT)
8/21RBS-lysis1

Template:Kyoto/Notebook/Sep 3

From 2013.igem.org

Contents

Sep 3

Electrophoresis

Miniprep

Liquid Culture

Restriction Enzyme Digestion

PCR

Mutatation PCR

Colony PCR

Electrophoresis

Electrophoresis

Gel Extraction

Liquid Culture