Template:Kyoto/Notebook/Sep 8

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Sep 8

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder
2	RBS-lysis1-DT	XbaI & PstI
3	RBS-lysis1-DT	XbaI & PstI
5	RBS-lysis3-DT	XbaI & PstI
6	RBS-lysis3-DT	XbaI & PstI

Name	concentration[µg/mL]	260/280	260/230
RBS-lysis1-DT (XbaI & PstI)	5.3	1.98	0.06
RBS-lysis3-DT (XbaI & PstI)	5.3	1.98	0.06

Lane	DNA	Enzyme
1	100bp ladder
3	Pcon-pT181 attenuator	EcoRI & SpeI
4	Pcon-pT181 attenuator	EcoRI & SpeI
6	Spinach-DT	XbaI & PstI
7	Spinach-DT	XbaI & PstI
9	RBS-lacZα-DT	XbaI & PstI
10	RBS-lacZα-DT	XbaI & PstI

Name	concentration[µg/mL]	260/280	260/230
Pcon-pT181 attenuator (EcoRI & SpeI)	5.2	1.82	0.36
Spinach-DT (XbaI & PstI)	8.0	1.87	0.25
RBS-lacZα-DT (XbaI & PstI)	10.3	1.71	0.54

Lane	DNA	Enzyme
1	1kbp ladder
2	Ptet-pT181 antisense	EcoRI & PstI
3	Ptet-pT181 antisense	EcoRI & PstI
5	pSB4K5	EcoRI & SpeI
6	pSB4K5	EcoRI & SpeI

Name	concentration[µg/mL]	260/280	260/230
Ptet-pT181 antisense(EcoRI & PstI)	30.6	1.84	1.16
pSB4K5 (EcoRI & SpeI)	21.8	1.87	0.98

Lane	DNA	Enzyme
1	1kbp ladder
2	Pcon-pT181 attenuator	SpeI & PstI
3	Pcon-pT181 attenuator	SpeI & PstI
5	Plac	SpeI & PstI
6	Plac	SpeI & PstI
8	pSB1C3	EcoRI & SpeI
9	pSB1C3	EcoRI & SpeI
11	pSB1C3	XbaI & PstI
12	pSB1C3	XbaI & PstI

Name	concentration[µg/mL]	260/280	260/230
Pcon-pT181 attenuator (SpeI & PstI)	14.5	1.91	1.09
Plac (SpeI & PstI)	11.6	1.74	1.19
pSB1C3 (EcoRI & SpeI)	5.7	1.74	0.34
pSB1C3 (XbaI & PstI)	5.6	2.00	0.37

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
Pcon-pT181 anti	243.0	1.90	1.81
Pcon-Spinach-DT	253.7	1.57	1.51

Ligation

Kojima

state	Vector(µL)		Inserter(µL)
experiment	9/8 Plac S+P	4.3	9/3 pT181 attenuator	3.1
experiment	9/8 Plac S+P	4.3	9/7 aptamer12-1R-DT X+P	2.1
experiment	9/8 Plac S+P	4.3	9/8 spinach-DT X+P	4.6
experiment	9/6 Plac S+P	2	9/7 RBS-lysis2-DT X+P	10
experiment	9/8 Plac S+P	4.3	9/7 RBS-lysis2-DT X+P	10
experiment	9/8 Ptet-pT181 anti	1.6	9/8 spinach-DT X+P	4.3
experiment	9/6 Pcon S+P	1.4	9/8 RBS-laczα-DT X+P	4.8
experiment	9/8 Pcon-pT181 attenuator S+P	3.4	9/7 aptamer12-1R-DT X+P	1.9
experiment	9/6 Pcon S+P	1.4	9/7 aptamer12-1R-DT X+P	2.1
experiment	9/8 DT	3.0	9/8 Pcon-pT181 attenuator E+S	7.9
experiment	9/6 Plux S+P	2	9/7 RBS-lysis1-DT X+P	7.2
experiment	9/8 Plac S+P	4.3	9/7 RBS-lysis1-DT X+P	7.2
experiment	9/4 Plac S+P	1.7	9/8 RBS-laczα-DT X+P	4.8

Restriction Enzyme Digestion

じっけんしゃ

	9/8 Pcon-pT181 anti	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	8.2µL	0µL	1µL	0µL	1µL	3µL	3µL	13.8µL	30µL
NC	0.4µL	0µL	0µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/8 Pcon-spin-DT	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	7.9µL	1µL	1µL	0µL	0µL	3µL	3µL	14.1µL	30µL
NC	0.4µL	0µL	0µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	8/21 F1 atte	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	6.5µL	0µL	0µL	1µL	1µL	3µL	3µL	15.5µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	8/21 F3m2 atte	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	6.0µL	0µL	0µL	1µL	1µL	3µL	3µL	16µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	8/21 F1 anti	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	6.7µL	0µL	0µL	1µL	1µL	3µL	3µL	15.3µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	8/21 F6 anti	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	6.0µL	0µL	0µL	1µL	1µL	3µL	3µL	16µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	8/21 tetR aptamer 12_1M	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	5.3µL	1µL	1µL	0µL	0µL	3µL	3µL	16.7µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	8/21 tetR aptamer 12_P	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	13.3µL	1µL	1µL	0µL	0µL	3µL	3µL	8.7µL	30µL
NC	0.7µL	0µL	0µL	0µL	0µL	1µL	1µL	7.3µL	10µL

	9/4 spin-DT	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	5.4µL	0µL	0µL	1µL	1µL	3µL	3µL	16.6µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	9/7 RBS-lysis1-DT	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	10µL	0µL	0µL	1µL	1µL	3µL	3µL	12µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL

	9/6 RBS-lysis2-DT	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	13µL	0µL	0µL	1µL	1µL	3µL	3µL	9µL	30µL
NC	0.7µL	0µL	0µL	0µL	0µL	1µL	1µL	7.3µL	10µL

	9/7 RBS-lysis3-DT	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	10µL	0µL	0µL	1µL	1µL	3µL	3µL	12µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL

	8/20 J23100 RBS-lacZα-DT-(1)	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	7µL	0.5µL	0.5µL	0µL	0µL	3µL	3µL	16µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL

	8/20 RBS-lacZα-DT	E	S	X	P	BSA	Buffer	MilliQ	total
2 cuts	10µL	0.5µL	0.5µL	0µL	0µL	3µL	3µL	13µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL

Transformation

じっけんしゃ

Name	Sample(µL)	Competent Cells(µL)	Total(µL)	Plate
9/8 Pcont+aptamer 12-1R-DT	1	10	11	Amp
9/8 Pcon+RBS-lacZα-DT	1	10	11	Amp
9/8 Pcon-pT181 attenuator+aptamer 12-1R-DT	1	10	11	Amp
9/8 Plac+RBS-lysis1-DT	2	20	22	CP
9/8 Plac+RBS-lysis2-DT	2	20	22	CP
9/8 Plac+aptamer 12-1R-DT	2	20	22	CP
9/8 Plac+spinach	2	20	22	CP
9/8 Plac+pT181 attenuator	2	20	22	CP
9/8 Plux+RBS-lysis1-DT	1	10	11	CP
9/8 Plux+RBS-lysis2-DT	1	10	11	CP
9/8 Ptet+RBS-lacZα-DT	1	10	11	CP
9/8 Ptet-pT181 anti+spinach-DT	1	10	11	CP
9/8 Pcon-pT181 attenuator+DT	1	10	11	CP

Electrophoresis

じっけんしゃ

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder
2	Pcon-spin-DT	E	S
3	Pcon-spin-DT
4	Fusion1 attenuator	X	P
5	Fusion1 attenuator
6	Fusion3m2 attenuator	X	P
7	Fusion3m2 attenuator
8	Fusion1 anti	X	P
9	Fusion1 anti
10	Fusion6 anti	X	P
11	Fusion6 anti
12	aptamer 12-1M	X	P
13	aptamer 12-1M
14	aptamer 12-P	X	P
15	aptamer 12-P
16	spinach-DT	X	P

Lane	Sample	Enzyme1	Enzyme2
1	spinach-DT
2	J23100-RBS-lacZα-DT	E	S
3	J23100-RBS-lacZα-DT
4	100bp ladder
5	RBS-lacZα-DT	E	S
6	RBS-lacZα-DT
7	RBS-lysis1-DT	X	P
8	RBS-lysis1-DT

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder
2	RBS-lysis2-DT	X	P
3	RBS-lysis2-DT
4	RBS-lysis3-DT	X	P
5	RBS-lysis3-DT
6	Pcon pT181 anti	S	P
7	Pcon pT181 anti

Gel Extraction

じっけんしゃ

Lane	DNA	Enzyme
1	100bp ladder
2	Pcon-spin-DT	E+S
3	Pcon-spin-DT	E+S
5	Fusion 1 attenuator	X+P
6	Fusion 1 attenuator	X+P
8	Fusion3m2 attenuator	X+P
9	Fusion3m2 attenuator	X+P
11	Fusion 1 anti	X+P
12	Fusion 1 anti	X+P

Name	concentration[µg/mL]	260/280	260/230
Pcon-spin-DT(E+S)	10.5	1.86	0.39
Fusion 1 attenuator(X+P)	7.0	1.71	0.27
Fusion3m2 attenuator(X+P)	8.6	1.75	0.47
Fusion 1 anti(X+P)	7.6	1.87	0.43

Lane	DNA	Enzyme
1	100bp ladder
2	Fusion6 anti	X+P
3	Fusion6 anti	X+P
5	aptamer12_1M	X+P
6	aptamer12_1M	X+P

Name	quantity	concentration[µg/mL]	260/280	260/230
Fusion6 anti(X+P)		5.0	1.76	0.20
aptamer12_1M(X+P)		3.3	2.31	0.04

Lane	DNA	Enzyme
1	100bp ladder
2	aptamer12_P	X+P
3	aptamer12_P	X+P
5	spinach-DT	E+S
6	spinach-DT	E+S

Name	quantity	concentration[µg/mL]	260/280	260/230
aptamer12_P(X+P)		3.4	1.72	0.26
spinach-DT(E+S)		10.1	1.57	0.47

Lane	DNA	Enzyme
1	100bp ladder
2	J23100-RBS-laczα-DT	E+S
3	J23100-RBS-laczα-DT	E+S
5	RBS-laczα-DT	E+S
6	RBS-laczα-DT	E+S
8	RBS-lysis1-DT	X+P
9	RBS-lysis1-DT	X+P
11	RBS-lysis2-DT	X+P
12	RBS-lysis2-DT	X+P

Name	concentration[µg/mL]	260/280	260/230
Pcon-RBS-laczα-DT(E+S)	5.6	2.03	0.25
RBS-laczα-DT(E+S)	4.4	2.04	0.33
RBS-lysis1-DT(X+P)	5.9	2.22	0.53
RBS-lysis2-DT(X+P)	5.1	1.67	0.48

Lane	DNA	Enzyme
1	100bp ladder
2	RBS-lysis3-DT	X+P
3	RBS-lysis3-DT	X+P
5	Pcon-pT181 anti	S+P
6	Pcon-pT181 anti	S+P

Name	quantity	concentration[µg/mL]	260/280	260/230
RBS-lysis3-DT(X+P)		23.4	1.72	0.88
Pcon-pT181 anti(S+P)		31.3	1.86	0.92

Ligation

じっけんしゃ

state	Vector		Inserter
experiment	Pcon-spin-DT(ES)	10.5µg/mL	pSB6A1(ES)	8.9µg/mL
experiment	Pcon-spin-DT(ES)	10.5µg/mL	pSB4K5(ES)	21.8µg/mL
experiment	Pcon-pT181 anti(S+P)	31.3µg/mL	spin-DT(X+P)	10.1µg/mL
experiment	Plac(S+P)	11.6µg/mL	RBS-lysis3-DT	23.4µg/mL
experiment	Plux(S+P)	25µg/mL	RBS-lysis3-DT	23.4µg/mL
experiment	Ptet-pT181 anti(S+P)	30.6µg/mL	spin-DT(X+P)	10.1µg/mL
experiment	Pcon-pT181 attenuator(S+P)	14.5µg/mL	RBS-laczα-DT(X+P)	4.4µg/mL

PCR

No name

KaiA(plasmid)	KOD plus	10x buffer	dNTP	MgSO4	Primer(661-RBS-KaiA-fwd)	Primer(kaiA-662-rev-ver2)	MilliQ	total
1	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	28s	30cycles

KaiB(plasmid)	KOD plus	10x buffer	dNTP	MgSO4	Primer(662-RBS-KaiB-fwd-ver2)	Primer(kaiB-663-rev-ver2)	MilliQ	total
1	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	28s	30cycles

DT(plasmid)	KOD plus	10x buffer	dNTP	MgSO4	Primer(664-DT-fwd-ver2)	Primer(DT-suffix-660-rev-ver2)	MilliQ	total
0.1	0.5	2.5	2.5	1.5	0.75	0.75	16.4	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	28s	30cycles

P-KaiB(PCR prpduct)	KOD plus	10x buffer	dNTP	MgSO4	Primer(prefix-PkaiBC-fwd-ver2)	Primer(PkaiBC-suffix--662-rev-ver2)	MilliQ	total
0.7	0.5	2.5	2.5	1.5	0.75	0.75	15.8	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	28s	30cycles

Plac(1)(plasmid)	KOD plus	10x buffer	dNTP	MgSO4	Primer(GG0-pSBIC3-fwd-ver2)	Primer(Plac-GG1-rev-ver2)	MilliQ	total
0.13	0.5	2.5	2.5	1.5	0.75	0.75	16.37	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	60s	30cycles

KaiC(plasmid)	KOD plus	10x buffer	dNTP	MgSO4	Primer(GG3-RBS-KaiC-fwd-ver2)	Primer(KaiC-GG4-rev-ver2)	MilliQ	total
1	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	60s	30cycles

Gel Extraction

じっけんしゃ

Lane	DNA	Enzyme
1	100bp ladder
2	KaiA
3	KaiA
4	KaiB
5	KaiB
6	DT
7	DT
8	P-KaiBC
9	P-KaiBC
10	nega
11	100bp ladder

Lane	DNA	Enzyme
1	100bp ladder
2	KaiA
3	KaiA
4	KaiB
5	KaiB
6	DT
7	DT
8	P-KaiBC
9	P-KaiBC
10	nega
11	100bp ladder

Lane	DNA	Enzyme
1	Plac
2	Plac
3	KaiC
4	KaiC
5	nega
6	1kb ladder

Template:Kyoto/Notebook/Sep 8

From 2013.igem.org

Contents

Sep 8

Gel Extraction

Miniprep

Ligation

Restriction Enzyme Digestion

Transformation

Electrophoresis

Gel Extraction

Ligation

PCR

Gel Extraction