Template:Kyoto/Notebook/DMS/23

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2/10

Restriction Enzyme Digestion

Yasuda

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
J23100	5.7	-	-	0.5	0.5	10xBuffer(K)	3	-	20.3	30
RBS-GFP-DT	7.5	-	0.5	-	0.5	10xBuffer(M)	3	3	15.5	30

Electrophoresis/Gel Extraction

Yasuda

Lane	Sample
1	1kbp ladder(2µL)
2	J23100 DNA control
3	J23100 S,P experimental
4	J23100 S,P experimental

Lane	Sample
1	1kbp ladder(2µL)
2	RBS-GFP-DT DNA control
3	RBS-GFP-DT X,P experimental
4	RBS-GFP-DT X,P experimental

Restriction Enzyme Digestion

Yasuda

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
J23100	10	-	-	0.5	0.5	10xBuffer(K)	3	-	16	30

2/12

Electrophoresis/Gel Extraction

Yasuda

Lane	Sample
1	1kbp ladder(2µL)
2	J23100 DNA control
3	J23100 S,P experimental
4	J23100 S,P experimental

Sample	Concentration/(µg/mL)	260/280	260/230
GFP	16.7	1.46	0.64
Pcon	17.2	1.69	0.26

Ligation

Inoue

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
2/10 J23100 S,P 2100bp	5.8	2/10 RBS-GFP-DT 898bp	4.9	2x Buffer	5	T4 Ligase	3	-	10

Transformation

Yasuda

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
2/12 J23119(A) Pcon	1	Self-Making Competent Cells (DH5α)	10	11	SOC
2/12 I712074 (Ak) T7	1	Self-Making Competent Cells (DH5α)	10	11	SOC
2/12 Ligation Pcon+RBS-GFP-PT	1	Self-Making Competent Cells (DH5α)	10	11	SOC

2/13

Colony PCR

Yasuda,Murakami

Quick Taq/(µL)	VF/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
30	1.2	1.2	27.6	60

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
5min	30sec	30sec	72sec

Denature-Annealing-Extension 30cycle

Number	Sample
1	Pcon-RBS-GFP-DT (1)
2	Pcon-RBS-GFP-DT (2)
3	Pcon-RBS-GFP-DT (3)
4	Pcon-RBS-GFP-DT (4)
5	T7

Electrophoresis

Honda

Lane	Sample
1	1kbp ladder
2	2/12 Pcon-RBS-GFP-DT (1)
3	2/12 Pcon-RBS-GFP-DT (2)
4	2/12 Pcon-RBS-GFP-DT (3)
5	2/12 Pcon-RBS-GFP-DT (4)
6	2/12 I712074(Ak) T7

Liquid Culture

No Name

Sample	Medium
Pcon-RBS-GFP-DT	Amp

2/14

Miniprep

Murakami

Sample
2/13 Pcon-RBS-GFP-DT(1)
2/13 Pcon-RBS-GFP-DT(2)

2/23

Self-Making Competent Cells (DH5α)

Tatsui and Kojima

3/4

Restriction Enzyme Digestion

Kato, Murata and Shimazaki

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
9/8 J23100 325µg/ml	6	-	-	0.5	0.5	H	3	3	17	30
9/8 J23100 325µg/ml	0.3	-	-	-	-	H	1	1	7.7	10
9/28 RBS-GFP-DT 215.9µg/ml	9.3	-	0.5	-	0.5	H	3	3	13.7	30
control	0.5	-	-	-	-	H	1	1	7.5	10

Electrophoresis

Kato, Murata and Shimazaki

Lane	Sample
1	1kbp ladder
2	9/8 J23100 S,P
3	9/8 J23100 control
4	9/28 RBS-GFP DT X,P
5	9/28 RBS-GFP DT control

Gel Extraction

Kato, Murata and Shimazaki

Lane	Sample
1	9/8 J23100 S,P
2	Blank
3	Blank
4	9/28 RBS-GFP-DT X,P
5	1kbp ladder

3/5

Ligation

Kato, Shimazaki and Murata

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
3/4 J23100 S,P	4.8	3/4 RBS-GFP-DT X,P	9.2	Reaction Buffer	2	Ligase(Bio Lub)	1	3	20

Transformation

No Name

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
3/5 J23119	1	Self-Making Competent Cells (DH5α)	10	11	SOC
3/5 I712074	1	Self-Making Competent Cells (DH5α)	10	11	SOC
3/5 ligation Pcon RBS-GFP-DT	1	Self-Making Competent Cells (DH5α)	10	11	SOC

3/6

Colony PCR

Kato and Shimazaki

Quick Taq/(µL)	VF/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
65	2.6	2.6	59.8	130

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
5min	30sec	30sec	72sec

Denature-Annealing-Extension 30cycle

Number	Sample
1	3/5 J23119 (1)
2	3/5 J23119 (2)
3	3/5 J23119 (3)
4	3/5 J23119 (4)
5	3/5 I712074 (1)
6	3/5 I712074 (2)
7	3/5 I712074 (3)
8	3/5 I712074 (4)

Number	Sample
1	3/5 Pcon RBS-GFP-DT (1)
2	3/5 Pcon RBS-GFP-DT (2)
3	3/5 Pcon RBS-GFP-DT (3)
4	3/5 Pcon RBS-GFP-DT (4)

Electrophoresis

Murata

Lane	Sample
1	1kbp ladder
2	3/5 J23119 (1)
3	3/5 J23119 (2)
4	3/5 J23119 (3)
5	3/5 J23119 (4)
6	3/5 I712074 (1)
7	3/5 I712074 (2)
8	3/5 I712074 (3)
9	3/5 I712074 (4)
10	3/5 Pcon RBS-GFP-DT (1)
11	3/5 Pcon RBS-GFP-DT (2)
12	3/5 Pcon RBS-GFP-DT (3)
13	3/5 Pcon RBS-GFP-DT (4)
14	1kbp ladder

3/7

Miniprep

Murata

Sample
3/6 Pcon RBS-GFP-DT (1)
3/6 Pcon RBS-GFP-DT (2)

3/10

Transformation

Okazaki

Sample/(µL)	CompetentCells/(µL)	Medium
3/10 RBS-GFP	Self-Making Competent Cells (DH5α)	CP
RBS	Self-Making Competent Cells (DH5α)	Amp
DT	Self-Making Competent Cells (DH5α)	CP
3/10 T7	Self-Making Competent Cells (DH5α)	Amp

3/11

Colony PCR

Shimazaki

Master Mix

Quick Taq/(µL)	VF/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
50	2	2	46	100

Predenature	Denature	Annealing	Extension
97°C	94°C	55°C	68°C
5min	30sec	30sec	60sec

Denature-Annealing-Extension 30cycle

Number	Sample
1	3/10 RBS (1)
2	3/10 RBS (2)
3	3/10 pSB1C3 (1)
4	3/10 pSB1C3 (2)
5	3/10 DT (1)
6	3/10 DT (2)
7	3/10 T7 (1)
8	3/10 T7 (2)
9	control

3/12

Electrophoresis

Shimazaki

Lane	Sample
1	3/10 RBS (1)
2	3/10 RBS (2)
3	3/10 pSB1C3 (1)
4	3/10 pSB1C3 (2)
5	3/10 DT (1)
6	3/10 DT (2)
7	3/10 T7 (1)
8	3/10 T7 (2)
9	control
10	1kbp ladder

Colony PCR

Shimazaki

Master Mix

Quick Taq/(µL)	VF/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
50	2.0	2.0	46	100

Predenature	Denature	Annealing	Extension
97°C	94°C	55°C	68°C
5min	30sec	30sec	60sec

Denature-Annealing-Extension 30cycle

Number	Sample
1	3/10 RBS 3
2	3/10 RBS 4
3	3/10 pSB1C3 4
4	3/10 pSB1C3 5
5	3/10 DT 4
6	3/10 DT 5
7	3/10 T7 3
8	3/10 T7 4
9	new QuickTaq control
10	old QuickTaq control

3/14

Electrophoresis

Kojima

Lane	Sample
1	1kbp ladder
2	3/10 RBS (1)
3	3/10 RBS (2)
4	3/10 pSB1C3 (3)
5	3/10 pSB1C3 (4)
6	3/10 DT (5)
7	3/10 DT (6)
8	3/10 T7 (7)
9	3/10 T7 (8)
10	old Quick Taq control (9)
11	new Quick Taq control (10)
12	1kbp ladder

Colony PCR

Kojima

Master Mix

Quick Taq/(µL)	VF/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
17.5	0.7	0.7	16.1	35

Predenature	Denature	Annealing	Extension
97°C	95°C	58°C	68°C
5min	30sec	30sec	30sec

Denature-Annealing-Extension 30cycle

Number	Sample
1	RBS-4
2	DT-5
3	control

Electrophoresis

Kojima

Lane	Sample
1	1kbp ladder
2	RBS-4
3	DT-5
4	control

Colony PCR

Yasuda and Shimazaki

Master Mix

Quick Taq/(µL)	VF/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
17.5	0.7	0.7	16.1	35

Predenature	Denature	Annealing	Extension
97°C	95°C	58°C	68°C
5min	30sec	30sec	60sec

Denature-Annealing-Extension 30cycle

Number	Sample
1	pSB1C3-3
2	T7-5
3	control

Electrophoresis

Shimazaki

Lane	Sample
1	1kbp ladder
2	pSB1C3-3
3	T7-5
4	control
4	2/10 MilliQ

3/15

Liquid Culture

Yasuda and Murata

Sample	Medium
Ruegeria pomeroyi fungus liquid	YTSS medium

3/17

Liquid Culture

Yoshida

Sample	Medium
3/10 pSB1C3-3	CP
3/10 DT-5	CP
3/10 T7-5	Amp
3/10 RBS-4	Amp

3/18

Miniprep

Murata and Yasuda

Sample	Concentration/(µg/mL)	260/280	260/230
3/17 DT CP	64.4	1.17	0.29
3/17 RBS Amp	61.4	1.28	0.36
3/17 T7 Amp	66.8	1.22	0.34
3/17 pSB1C3	64.2	1.31	0.39

Restriction Enzyme Digestion

Yoshida

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
pSB1C3 E,S 3/18 miniprep product	20	1	1	-	-	M	3	3	2	30
control	3	-	-	-	-	M	1	1	5	10

Cloning PCR

Murata and Yasuda

2K KAPA HiFi HotStart ReadyMix/(µL)	F dddD 66/(µL)	R dddD 65/(µL)	R.pomeroyi Genome/(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	3	8	25

Predenature	Denature	Annealing	Extension	Final Extension
95°C	98°C	65°C	72°C	72°C
3min	20sec	15sec	150sec	1sec

Denature-Annealing-Extension 25cycle

3/19

Electrophoresis

Yoshida

Lane	Sample
1	1kbp ladder
2	pSB1C3 E,X
3	pSB1C3 control

Restriction Enzyme Digestion

Yoshida

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
pSB1C3 3/18 miniprep product	20	1	-	1	-	H	3	-	5	30
control	3	-	-	-	-	H	1	-	6	10

Electrophoresis

Kojima

Lane	Sample
1	1kbp ladder
2	dddD PCR product

PCR

Kojima

3/19 DNA Purification product/(µL)	Fw/(µL)	Rv/(µL)	HiFi Mix/(µL)	MilliQ/(µL)	Total/(µL)
1	0.75	0.75	12.5	10	25

R.pomeroyi Genome/(µL)	Fw/(µL)	Rv/(µL)	HiFi Mix/(µL)	MilliQ/(µL)	Total/(µL)
3	0.75	0.75	12.5	8	25

Predenature	Denature	Annealing	Extension	Final Extension
95°C	98°C	68°C	72°C	72°C
5min	20sec	15sec	90sec	5min

Denature-Annealing-Extension 25cycle

3/20

Electrophoresis

Kojima

Lane	Sample
1	1kbp ladder
2	pSB1C3 E,S
3	control
4	dddD(1) 3/19 PCR product
5	dddD(2) 3/19 PCR product

Gel Extraction

Yasuda and Tatsui

Lane	Sample
1	Blank
2	1kbp ladder
3	pSB1C3
4	Blank
5	pSB1C3

Gel Extraction

Murara

Lane	Sample
1	1kbp ladder
2	dddD
3	dddD

Sample	Concentration/(µg/mL)	260/280	260/230
dddD	35	1.63	0.85

3/24

Restriction Enzyme Digestion

Honda

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
pSB1C3	11	0.5	0.5	-	-	M	1.5	1.5	-	15
dddD	12.5	0.5	-	0.5	-	M	1.5	-	-	15

PCR

Honda

2x KAPA HiFi HotStart ReadyMix/(µL)	F dddD 62/(µL)	R dddD 68/(µL)	Templete 3/24 dddD/(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	pick up	11	25

Predenature	Denature	Annealing	Extension	Final Extension
95°C	98°C	68°C	72°C	72°C
5min	20sec	15sec	90sec	5min

Denature-Annealing-Extension 25cycle

Number	Sample
1	dddD

Sample	Concentration/(µg/mL)	260/280	260/230
dddD	62.7	1.77	1.88

Ligation

Honda

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
dddD	1.3	pSB1C3	0.43	2xBuffer	5	T4Ligase	1	2.27	10

Liquid Culture

Tatsui

Sample	Medium
pSB1C3(3)	CP
pSB1C3(4)	CP

3/25

Miniprep

Tatsui

Sample	Concentration/(µg/mL)
pSB1C3 3/24 (3)	41
pSB1C3 3/24 (4)	57

Restriction Enzyme Digestion

Murata

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
pSB1C3 3/24 (3)	15.4	0.5	-	0.5	-	H	3	-	10.6	30
3/24 PCR product dddD E,S	3.2	0.5	-	0.5	-	H	3	-	22.8	30

Gel Extraction

Murata

Lane	Sample
1	1kbp ladder
2	pSB1C3(3) E,S 3/25
3	Blank
4	Blank
5	Blank
6	dddD 3/25 E,S
7	dddD 3/25 E,S

Sample	Concentration/(µg/mL)	260/280	260/230
dddD	4.9	17.5	0.52
pSB1C3	2.0	-	0.21

3/26

Ligation

Yasuda

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
pSB1C3 2047bp	30	dddD 2545bp	30	2x Rapid Ligation Buffer	5	T4Ligase	3	-	10

Transformation

Yasuda

Sample/(µL)
dddD(pSB1C3)

3/27

Restriction Enzyme Digestion

Murata

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
pSB1C3(4) 3/25	20.1	0.5	-	0.5	-	10xBuffer	3	-	5.9	30
dddD 3/24 PCR product	3.2	0.5	-	0.5	-	10xBuffer	3	-	22.8	30

Gel Extraction

Honda

Lane	Sample
1	1kbp ladder
2	pSB1C3 E,S 3/27
3	pSB1C3 E,S 3/27
4	Blank
5	dddD E,S 3/27
6	dddD E,S 3/27

Sample	Concentration/(µg/mL)	260/280	260/230
pSB1C3	9	1.58	0.04
dddD	16.0	1.78	0.08

Ligation

Honda

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
pSB1C3	11	dddD	15	2x Rapid Ligation Buffer	5	T4 DNA Ligase	1	4	10

3/28

PCR

Yasuda

2x KAPA HiFi HotStart ReadyMix/(µL)	F Sequence primer/(µL)	R Spe1-dddD/(µL)	Templete/(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	pick up	11	25

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
95°C	98°C	68°C	72°C	72°C
5min	20sec	15sec	90sec	5min

Denature-Annealing-Extension 25cycle

Number	Sample
1	Fw:Sequence primer (1) 57.9/Rv:Spe1 dddD 2276bp
2	Fw:Sequence primer (2) 57.3/Rv:Spe1 dddD 1907bp
3	Fw:Sequence primer (3) 57.6/Rv:Spe1 dddD 1527bp
4	Fw:Sequence primer (4) 56.3/Rv:Spe1 dddD 1189bp
5	Fw:Sequence primer (5) 57.2/Rv:Spe1 dddD 799bp
6	Fw:Sequence primer (6) 56.4/Rv:Spe1 dddD 398bp
7	Fw:Sequence primer VF2 56.4/Rv:Spe1 dddD 266bp
8	Fw:pre-dddD PCR product 62/Rv:Spe1 dddD
9	Fw:Blank/Rv:Spe1 dddD

Electrophoresis

Murata

Lane	Sample
1	1kbp ladder
2	Seaquence primer (1) PCR product
3	Seaquence primer (2) PCR product
4	Seaquence primer (3) PCR product
5	Seaquence primer (4) PCR product
6	Seaquence primer (5) PCR product
7	Seaquence primer (6) PCR product
8	Seaquence primer VF2
9	F pre-dddD cloning
10	Blank
11	1kbp ladder

Transformation

Kojima

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
dddD	1	Self-Making Competent Cells (DH5α)	10	11	CP

3/30

Colony PCR

Yasuda

Quick Taq/(µL)	VF/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
5	0.2	0.2	4.6	10

Predenature	Denature	Annealing	Extension
97°C	95°C	58°C	68°C
5min	30sec	30sec	168sec

Denature-Annealing-Extension 30cycle

Number	Sample
1	3/26 dddD(pSB1C3) (1)
2	3/26 dddD(pSB1C3) (2)
3	3/26 dddD(pSB1C3) (3)
4	3/26 dddD(pSB1C3) (4)
5	3/26 dddD(pSB1C3) (5)
6	3/28 dddD(pSB1C3) (6)
7	3/28 dddD(pSB1C3) (7)
8	3/28 dddD(pSB1C3) (8)
9	3/28 dddD(pSB1C3) (9)
10	3/28 dddD(pSB1C3) (10)

3/31

Electrophoresis

Yasuda

Number	Sample
1	1kbp ladder
2	3/26 dddD(pSB1C3) (1)
3	3/26 dddD(pSB1C3) (2)
4	3/26 dddD(pSB1C3) (3)
5	3/26 dddD(pSB1C3) (4)
6	3/26 dddD(pSB1C3) (5)
7	1kbp ladder
8	3/28 dddD(pSB1C3) (6)
9	3/28 dddD(pSB1C3) (7)
10	3/28 dddD(pSB1C3) (8)
11	3/28 dddD(pSB1C3) (9)
12	3/28 dddD(pSB1C3) (10)
13	1kbp ladder