Template:Kyoto/Notebook/DMS

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7/31

Restriction Enzyme Digestion

Nakamura and Matsumoto

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
3/24 dddD PCR product	3.2	1	-	1	-	H	3	-	21.8	30
pSB1C3	18.5	1	-	1	-	H	3	-	6.5	30
pSB1C3 control	6.2	-	-	-	-	H	1	-	2.8	10

8/1

Gel Extraction

Murata and Yasuda

Lane	Sample
1	1kbp ladder
2	3/24 dddD PCR product E,S
3	3/24 dddD PCR product E,S
4	Blank
5	pSB1C3 E,S
6	pSB1C3 E,S
7	Blank
8	1kbp ladder
9	3/24 dddD PCR product control
10	pSB1C3 control

Sample	Concentration/(µg/mL)	260/280	260/230
PSB1C3	13.3	1.90	-
dddD	10.8	1.64	0.15

Ligation

Yasuda

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
8/1 PSB1C3 gel extraction product 2047bp	1.5	8/1 dddD gel extraction product 2545bp	6.9	10xT4Ligase	2	T4Ligase	1	8.6	20

Transformation

Murata

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
PSB1C3-dddD	3	Self-Making Competent Cells (DH5α)	30	33	SOC

8/2

PCR Cloning

Murata

dddD without Pre-Suf	2x KAPA HiFi Hotstart Ready Mix/(µL)	F Pre-dddD PCR product 62/(µL)	Rv Spe1-dddD PCR product 68/(µL)	MilliQ/(µL)	Total/(µL)
pick up	12.5	0.75	0.75	11	25

Predenature	Denature	Annealing	Extension
95°C	98°C	65°C	72°C
5min	20sec	15sec	50sec

Denature-Annealing-Extension 25cycle

DNA Purification

Murata

8/2 Pre-dddD-Suf PCR product

Colony PCR

Honda,Nakamura and Matsumoto

Master Mix

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
15	0.6	0.6	13.8	30

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	24sec

Denature-Annealing-Extension 30cycle

Number	Sample
1	pSB1C3-dddD(1)
2	pSB1C3-dddD(2)
3	pSB1C3-dddD(3)

Concentration Measurement

Nakamura and Matsumoto

Sample	Concentration/(µg/mL)	260/280	260/230
dddD PCR product	245	1.59	2.17

Restriction Enzyme Digestion

Nakamura and Matsumoto

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
8/2 dddD PCR product	8.2	1	-	1	-	H	3	-	16.8	30
8/2 dddD PCR product control	0.41	-	-	-	-	H	1	-	8.6	10
pSB1C3	12.7	1	-	1	-	H	3	-	12.3	30
pSB1C3 control	0.04	-	-	-	-	H	1	-	8.4	10

8/3

Electrophoresis

Murata

Lane	Sample
1	pSB1C3-dddD(1)
2	pSB1C3-dddD(2)
3	pSB1C3-dddD(3)
4	1kb ladder (3µL)
5	1kb ladder (1µL)

Gel Extraction

Tatsui

Lane	Sample
1	1kb ladder
2	Blank
3	pSB1C3 2070bp
4	Blank
5	pSB1C3
6	Blank
7	8/2 dddD PCR product 2544bp
8	Blank
9	8/2 dddD PCR product

Sample	Concentration/(µg/mL)	260/280	260/230
pSB1C3	12.5	1.37	0.42
dddD PCR product	35	1.54	0.85

8/4

Colony PCR

Honda

Master Mix

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
40	1.6	1.6	36.8	80

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	24sec

Denature-Annealing-Extension 30cycle

1	dddD+pSB1C3(1)-1
2	dddD+pSB1C3(1)-2
3	dddD+pSB1C3(2)-1
4	dddD+pSB1C3(2)-2
5	dddD+pSB1C3(2)-3
6	dddD+pSB1C3(2)-4
7	pSB1C3 control-1
8	control

Electrophoresis

Matsumoto

Lane	Sample
1	1kbp ladder
2	dddD+pSB1C3(1)-1
3	dddD+pSB1C3(1)-2
4	dddD+pSB1C3(2)-1
5	dddD+pSB1C3(2)-2
6	dddD+pSB1C3(2)-3
7	dddD+pSB1C3(2)-4
8	pSB1C3 control-1
9	control
10	1kbp ladder

PCR

Honda

Unexpected bands are confirmed in former Electrophoresis, so contamination of MilliQ or Quicktaq is suspected. For confirming contamination, PCR and Electrophoresis are conducted.

* suspicious samples for contamination

(1)

*Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
5	0.2	0.2	4.6	10

(2)

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
5	0.2	0.2	4.6	10

(3)

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	*MilliQ/(µL)	Total/(µL)
5	0.2	0.2	4.6	10

(4)

*Quick Taq/(µL)	VF2/(µL)	VR/(µL)	*MilliQ/(µL)	Total/(µL)
5	0.2	0.2	4.6	10

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	24sec

Denature-Annealing-Extension 30cycle

Electrophoresis

Honda

Lane	Sample
1	1kbp ladder
2	(1)
3	(2)
4	(3)
5	(4)

8/18

PCR

Honda

For confirming contamination of VF2 and VR primers, we pour samples into new tubes and perform PCR and Electrophoresis.

(1)

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
5	0.2	0.2	4.6	10

control(2)

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	8/2 Pcon3/(µL)	MilliQ/(µL)	Total/(µL)
5	0.2	0.2	0.6	4.6	10

(3)

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
5	0.2	0.2	4.6	10

(4)

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	8/2 Pcon3/(µL)	MilliQ/(µL)	Total/(µL)
5	0.2	0.2	0.6	4.0	10

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	24sec

Denature-Annealing-Extension 30cycle

Electrophoresis

Honda

Number	Sample
1	1kbp ladder
2	(1)
3	(2)
4	(3)
5	(4)

Restriction Enzyme Digestion

Honda

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
dddD 8/2 PCR product	5.5	0.7	-	0.7	-	H	2	-	11.1	20
dddD 8/2 PCR product control	0.41	-	-	-	-	H	1	-	8.6	10
pSB1C3	12.7	1	-	1	-	H	3	-	8.4	10
pSB1C3 control	0.64	-	-	-	-	H	1	-	8.4	10

Colony PCR

Honda

Master Mix

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
40	1.6	1.6	36.8	80

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	24sec

Denature-Annealing-Extension 30cycle

Number	Sample
1	dddD+pSB1C3(1)-1
2	dddD+pSB1C3(1)-2
3	dddD+pSB1C3(2)-1
4	dddD+pSB1C3(2)-2
5	dddD+pSB1C3(2)-3
6	dddD+pSB1C3(2)-4
7	pSB1C3 control-1
8	control

Electrophoresis

Honda

Lane	Sample
1	1kbp ladder
2	dddD+pSB1C3(1)-1
3	dddD+pSB1C3(1)-2
4	dddD+pSB1C3(2)-1
5	dddD+pSB1C3(2)-2
6	dddD+pSB1C3(2)-3
7	dddD+pSB1C3(2)-4
8	pSB1C3 control-1
9	control

Gel Extraction

Murata

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

Sample	Concentration/(µg/mL)	260/280	260/230
105	1.30	0.85
dddD	105	1.27	0.73

8/19

Ligation

Honda,Li,Yamauchi

(1)

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
pSB1C3	3.6	dddD	1	2xBuffer	5.6	T4Ligase	1	-	11.2

(2)

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
pSB1C3	3.6	-	-	2xBuffer	5.6	T4Ligase	1	1	11.2

8/20

Transformation

Murata

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
8/19 Ligation (1)	11.2	Competent Cells (DH5α)	50	61.2	SOC
8/19 Ligation (2)	11.2	Competent Cells (DH5α)	50	61.2	SOC

Ligation

Nakamura,Honda

(1)

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
pSB1C3	10.8	dddD	3	2xBuffer	16.8	T4Ligase	3	-	33.6

(2)

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
pSB1C3	10.8	-	-	2xBuffer	16.8	T4Ligase	3	3	33.6

Transformation

Nakamura,Honda

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
dddD+pSB1C3	5	Competent Cells (DH5α)	20	25	SOC
control	5	Competent Cells (DH5α)	20	25	SOC

8/21

Colony PCR

Honda,Nakamura

Master Mix

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
35	1.4	1.4	32.2	70

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	24sec

Denature-Annealing-Extension 30cycle

Electrophoresis

Nakamura

Number	Sample
1	8/20 dddD+pSB1C3(1)-1
2	8/20 dddD+pSB1C3(1)-2
3	8/20 dddD+pSB1C3(2)-1
4	8/20 dddD+pSB1C3(2)-2
5	8/20 dddD+pSB1C3(3)-1
6	8/20 dddD+pSB1C3(3)-2
7	control

Number	Sample
1	1kbp ladder
2	8/20 dddD+pSB1C3(1)-1
3	8/20 dddD+pSB1C3(1)-2
4	8/20 dddD+pSB1C3(2)-1
5	8/20 dddD+pSB1C3(2)-2
6	8/20 dddD+pSB1C3(3)-1
7	8/20 dddD+pSB1C3(3)-2
8	control

Gel Extraction

Yasuda

Lane	Sample
1	1kbp ladder
2	8/22 Mp(1) E,S
3	8/22 Mp(1) E,S
4	8/22 Mp(1) E,S

Lane	Sample
1	1kbp ladder
2	8/22 Mp(2) E,S
3	8/22 Mp(2) E,S
4	Blank
5	1kbp ladder
6	8/22 Mp(3) E,S
7	8/22 Mp(3) E,S

8/25

Gel Extraction

Yasuda

Lane	Sample
1	1kbp ladder
2	8/22 Mp(1) E,S
3	8/22 Mp(1) E,S
4	8/22 Mp(1) E,S

Ligation

Yasuda

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
8/22 Mp(1) E,S	1.3	8/18 dddD gel extraction product	0.81	2x Buffer	5	T4 Ligase	0.3	2.69	10
8/22 Mp(2) E+S	0.95	8/18 dddD gel extraction product	0.71	2x Buffer	5	T4 Ligase	0.3	3.04	10
8/22 Mp(3) E+S	0.69	8/18 dddD gel extraction product	0.71	2x Buffer	5	T4Ligase	0.3	3.3	10

8/26

Transformation

Nakamura,Matsumoto

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
8/25 Ligation (1)	10	Competent Cells (DH5α)	50	60	SOC
8/25 Ligation(2)	10	Competent Cells (DH5α)	50	60	SOC
8/25 Ligation(3)	10	Competent Cells (DH5α)	30	40	SOC

8/27

Colony PCR

Murata

Master Mix

Quick Taq/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
80	3.2	3.2	73.6	160

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	2h20min

Denature-Annealing-Extension 30cycle

Number	Sample
1	8/26 dddD+pSB1C3(1)-1
2	8/26 dddD+pSB1C3(1)-2
3	8/26 dddD+pSB1C3(1)-3
4	8/26 dddD+pSB1C3(1)-4
5	8/26 dddD+pSB1C3(1)-5
6	8/26 dddD+pSB1C3(2)-1
7	8/26 dddD+pSB1C3(2)-2
8	8/26 dddD+pSB1C3(2)-3
9	8/26 dddD+pSB1C3(2)-4
10	8/26 dddD+pSB1C3(2)-5
11	8/26 dddD+pSB1C3(3)-1
12	8/26 dddD+pSB1C3(3)-2
13	8/26 dddD+pSB1C3(3)-3
14	8/26 dddD+pSB1C3(3)-4
15	8/26 dddD+pSB1C3(3)-5
16	control

Electrophoresis

Yasuda

Lane	Sample
1	1kbp ladder
2	8/26 dddD+pSB1C3(1)-1
3	8/26 dddD+pSB1C3(1)-2
4	8/26 dddD+pSB1C3(1)-3
5	8/26 dddD+pSB1C3(1)-4
6	8/26 dddD+pSB1C3(1)-5

Lane	Sample
1	1kbp ladder
2	8/26 dddD+pSB1C3(2)-1
3	8/26 dddD+pSB1C3(2)-2
4	8/26 dddD+pSB1C3(2)-3
5	8/26 dddD+pSB1C3(2)-4
6	8/26 dddD+pSB1C3(2)-5
7	1kbp ladder
8	8/26 dddD+pSB1C3(3)-1
9	8/26 dddD+pSB1C3(3)-2
10	8/26 dddD+pSB1C3(3)-3
11	8/26 dddD+pSB1C3(3)-4
12	8/26 dddD+pSB1C3(3)-5
13	1kbp ladder
14	control

8/29

PCR

Matsumoto

Master Mix

2x KARA HiFi HotStart ReadyMix /(µL)	V Pre-dddD 62/(µL)	Rv Spe1-dddD 68/(µL)	MilliQ/(µL)	DNA Purification dddD without Pre-Suf	Total/(µL)
12.5	0.75	0.75	11	pick up	25

Predenature	Denature	Annealing	Extension
95°C	98°C	65°C	72°C
5min	20sec	15sec	50sec

Denature-Annealing-Extension 25cycle

DNA Purification

Matsumoto

Sample	Concentration/(µg/mL)	260/280	260/230
8/29 Pre-dddD-Suf PCR product	335	1.73	1.91

Restriction Enzyme Digestion

Matsumoto

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
8/29 Pre-dddD-Suf PCR product	6.0	1	-	1	-	H	3	-	19	30

DNA Purification

Matsumoto

Sample	Concentration/(µg/mL)	260/280	260/230
8/29 Pre-dddD-Suf Restriction Enzyme Digestion product	60	1.64	0.94

Ligation

Matsumoto

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
pSB1C3 E,S gel extraction product	4.7	dddD-pSB1C3	4	2x Buffer	9.7	T4 Ligase	1	-	19.4
pSB1C3 E,S gel extraction product	4.7	-	-	2x Buffer	9.7	T4 Ligase	1	4	19.4

Transformation

Matsumoto

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
dddD 8/29 ligation product	10	DH5a	40	50	SOC
8/29 ligation product control	10	DH5a	40	50	SOC
pSB1C3 control	1	DH5a	20	21	SOC

9/1

Colony PCR

Honda

Master Mix

Quick Tag/(µL)	VF2/(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
2min	30sec	30sec	24sec

Denature-Annealing-Extension 30cycle

Number	Sample
1	pSB1C3-dddD(1)
2	pSB1C3-dddD(2)
3	pSB1C3-dddD(3)
4	pSB4A5-(1)
5	pSB4A5-(2)
6	control

Electrophoresis

Matsumoto

Lane	Sample
1	1kb ladder
2	9/1 dddD+pSB1C3-1
3	9/1 dddD+pSB1C3-2
4	9/1 dddD+pSB1C3-3
5	9/1 pSB4A5-1
6	9/1 pSB4A5-2
7	control
8	1kb ladder

Colony PCR

Matsumoto

Master Mix

Quick Tag/(µL)	VF2/(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
2min	30sec	30sec	24sec

Denature-Annealing-Extension 30cycle

Number	Sample
1	pSB1C3-dddD(1)
2	pSB1C3-dddD(2)
3	pSB1C3-dddD(3)
4	pSB4A5-(1)
5	pSB4A5-(2)
6	control

Electrophoresis

Matsumoto

Lane	Sample
1	1kb ladder
2	9/1 dddD+pSB1C3-1
3	9/1 dddD+pSB1C3-2
4	9/1 dddD+pSB1C3-3
5	9/1 pSB4A5-1
6	9/1 pSB4A5-2
7	control
8	1kb ladder

9/2

Electrophoresis

Mastumoto

Lane	Sample
1	1kb ladder
2	8/29 dddD(DNA purification product)1µL+MilliQ 5µL+looding dye(x6)1µL
3	control

Cloning

Yasuda

2x KAPA HiFi HotStart ReadyMix/(µL) Fw REDOX cloning primer 59/(µL) Rv REDOX primer Ver.2 64/(µL) DNA CCAP 1023/1 /(µL) MilliQ/(µL) Total/(µL)

12.5 0.75 0.75 1 10 25

Predenature Denature Annealing Extension Final Extention Cooling

95°C 98°C 65°C 72°C 72°C 4°C

5min 20sec 15sec 2min10sec 5min long time

Denature-Annealing-Extension 25cycle

Restriction Enzyme Digestion

Matsumoto

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
8/29 dddD(DNA purification product)	6	1	-	1	-	-	3	-	19	30

Cloning

Yasuda
Master Mix(x4)

2x KAPA HiFi HotStart ReadyMix/(µL) Fw primer 59/(µL) Rv primer Ver.2 64/(µL) 3/19 dddD(DNA purification) /(µL) MilliQ/(µL) Total/(µL)

12.5 0.75 0.75 0.1 11 25

Predenature Denature Annealing Extension Final Extention Cooling

95°C 98°C 65°C 72°C 72°C 4°C

5min 20sec 15sec 2min10sec 5min long time

Denature-Annealing-Extension 25cycle

9/3

Gel Extraction

Yamauchi

Lane	Sample
2	dddD (E,S) 2544bp.
4	dddD (E,S)
6	1kb ladder

DNA Purification

Matsumoto

Sample	Concentration/(µg/mL)	260/280	260/230
Pre-dddD-suf	685	1.66	2.13

Ligation

Honda, Li and Yamauchi
(1)insert: dddD

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
pSB1C3	1.5	dddD	6.3	2x Buffer	8.8	T4 Ligase	1	-	17.6

(2)control

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
pSB1C3	1.5	dddD	-	2x Buffer	8.8	T4 Ligase	1	6.3	17.6

Transformation

Yamauchi

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
9/3 Ligation product (dddD)	3	DH5α	15	18	SOC
9/3 Ligation product (control)	2	DH5α	10	12	SOC

30min on ice
1min heat shock
2min on ice

(1)Adding SOC medium(4 times) to each of them and doing preculture at 37°C for 1 hour.
(2)Adding SOC medium to each of them, up to 100µL

9/4

Electrophoresis

Honda

Lane	Sample
1	1kb ladder
2	9/3 Ligation dddD + pSB1C3
3	8/20 Ligation dddD + pSB1C3
4	8/1 Ligation dddD + pSB1C3
5	9/3 dddD (Gel extraction product)(E,S)
6	9/3 dddD (DNA purification product)
7	1kb ladder

Cloning

Murata and Honda
(1)

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw primer AT cloning primer 61/(µL)	Rv primer AT cloning primer 61/(µL)	DNA CCAP 1023/1 /(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

(2)

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw SAMmt cloning primer 58/(µL)	Rv SAMmt cloning primer 57/(µL)	DNA CCAP 1023/1 /(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

(3)

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw DECARB cloning primer 56/(µL)	Rv DECARB cloning primer 56/(µL)	DNA CCAP 1023/1 /(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

(4)

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw DiDECARB cloning primer 57/(µL)	Rv DiDECARB cloning primer 57/(µL)	DNA CCAP 1023/1 /(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

(1)(4)

Predenature	Denature	Annealing	Extension	Final Extention	Cooling
98°C	98°C	60°C	72°C	72°C	4°C
5min	20sec	15sec	90sec	5min	long time

Denature-Annealing-Extension 25cycle

(2)(3)

Predenature	Denature	Annealing	Extension	Final Extention	Cooling
98°C	98°C	60°C	72°C	72°C	4°C
5min	20sec	15sec	70sec	5min	long time

Denature-Annealing-Extension 25cycle

Electrophoresis

Honda

Lane	Sample
1	1kb ladder
2	AT cloning(1)
3	SAMmt cloning(2)
4	DECARB cloning(3)
5	DiDECARB cloning(4)
6	1kb ladder

Restriction Enzyme Digestion

Yasuda
(1)

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
I732095 8/22 miniprep (2)(255ng/µL)	7.8	1	-	1	-	H(x10)	3	-	17.2	30
I732095 8/22 miniprep (2)(255ng/µL)	0.4	-	-	-	-	H(x10)	1	-	8.6	10

incubate at 37°C overnight
(2)

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
I732095 8/22 miniprep (2)(255ng/µL)	7.8	1	-	1	-	H(x10)	3	-	17.2	30
I732095 8/22 miniprep (2)(255ng/µL)	0.4	-	-	-	-	H(x10)	1	-	8.6	10
I732095 8/22 miniprep (1)(430ng/µL)	4.6	1	-	1	-	H(x10)	3	-	20.4	30

9/5

Gel Extraction

Honda and Yamauchi

Lane	Sample
1	1kb ladder
2	9/4 I732095(E,S)-1
3	-
4	9/4 I732095(E,S)-1
5	-
6	9/4 I732095(E,S)-1

Genome Cloning

Honda
(1)

2K KAPA HiFi HotStart ReadyMix/(µL)	Fw Primer AT cloning primer 61/(µL)	Rv Primer AT cloning primer 61/(µL)	DNA CCAP 1023/1 /(µL)	MilliQ/(µL)	Total/(µL)
50	3	3	4	40	100

(2)

2K KAPA HiFi HotStart ReadyMix/(µL)	Fw Primer SAMmt cloning primer 58/(µL)	Rv Primer SAMmt cloning primer 57/(µL)	DNA CCAP 1023/1/(µL)	MilliQ/(µL)	Total/(µL)
50	3	3	4	40	100

(3)

2K KAPA HiFi HotStart ReadyMix/(µL)	Fw Primer DiDECARB cloning primer 57/(µL)	Rv Primer DiDECARB cloning primer 57/(µL)	DNA CCAP 1023/1/(µL)	MilliQ/(µL)	Total/(µL)
50	3	3	4	40	100

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
98°C	98°C	55-65°C	72°C	72°C	12°C
3min	20sec	15sec	150sec	1sec	long time

Denature-Annealing-Extension 25cycle

Electrophoresis

Honda
(1)AT

Lane	Sample
1	-
2	-
3	-
4	1kb ladder
5	(1)-1
6	(1)-2
7	(1)-3
8	(1)-4
9	(1)-5
10	(1)-6
11	(1)-7
12	(1)-8
13	1kb ladder
14	-
15	-
16	-
17	-

(2)SAMmt

Lane	Sample
1	-
2	-
3	-
4	1kb ladder
5	(2)-1
6	(2)-2
7	(2)-3
8	(2)-4
9	(2)-5
10	(2)-6
11	-
12	-
13	-
14	-
15	-
16	(2)-8
17	1kb ladder

(3)DiDECARB

Lane	Sample
1	-
2	-
3	-
4	1kb ladder
5	(3)-1
6	(3)-2
7	(3)-3
8	(3)-4
9	(3)-5
10	(3)-6
11	(3)-7
12	(3)-8
13	1kb ladder
14	-
15	-
16	-
17	-

Gel Extraction

Murata

Lane	Sample
1	1kb ladder
2	9/4 I732045(E,S)
3	9/4 I732045(E,S)
4	-
5	-
6	9/4 I732045(E,S)
7	9/4 I732045(E,S)
8	-
9	1kb ladder
10	9/4 control

9/8

Genome Cloning and PCR

Honda and Yamaura
(1)

2K KAPA HiFi HotStart ReadyMix/(µL)	Fw Primer AT cloning primer 61/(µL)	Rv Primer AT cloning primer 61/(µL)	9/5 AT (genome cloning product) (1)-3 /(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

(2)

2K KAPA HiFi HotStart ReadyMix/(µL)	Fw Primer DiDECARB cloning primer 61/(µL)	Rv Primer DiDECARB cloning primer 61/(µL)	9/5 AT (genome cloning product) (3)-3 /(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

(3)

2K KAPA HiFi HotStart ReadyMix/(µL)	Fw Primer Pre-dddD cloning primer/(µL)	Rv Primer suf-dddD cloning primer/(µL)	3/19 dddD (DNA purification product)(121µg/µL)/(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
98°C	98°C	60°C	72°C	72°C	4°C
5min	20sec	15sec	90sec	5min	long time

Denature-Annealing-Extension 25cycle

Electrophoresis

Honda

Lane	Sample
1	1kb ladder
2	PCR product (AT)
3	PCR product (DIDECARB)
4	PCR product (dddD)
5	PCR product (dddD)
6	1kb ladder

DNA Purification

Matsumoto

Sample	Concentration/(µg/mL)	260/280	260/230
9/8 AT	55	1.37	1.14
9/8 DiDECARB	95	1.69	3.04
9/8 dddD	145	1.52	1.78

Restriction Enzyme Digestion

Matsumoto

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
9/8 dddD (DNA purificaion and PCR product)	13.8	1	-	1	-	H	3	-	11.2	30
control	0.7	-	-	-	-	H	1	-	8.3	10

Genome Cloning and PCR

Yasuda

2K KAPA HiFi HotStart ReadyMix/(µL)	Fw Primer SAMmt cloning primer 58/(µL)	Rv Primer SAMmt cloning primer 57/(µL)	9/5 SAMmt (genome cloning product) No.3/(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
98°C	98°C	63°C	72°C	72°C	12°C
5min	20sec	15sec	90sec	5min	long time

Denature-Annealing-Extension 25cycle

パーツ起こし@

Honda

9/9

カラム精製

Honda

Ligation

Li (1)Sample (2)Control

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
pSB1C3	4.2	dddD	3.8	(2x)Buffer	9	T4 Ligase	1	-	18
pSB1C3	4.2	-	-	(2x)Buffer	9	T4 Ligase	1	3.8	18

Transformation

Li

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
9/9 dddD+pSB1C3 ligation product	5	DH5α	50	55	SOC
9/9 dddD+pSB1C3 ligation product control	3	DH5α	30	33	SOC

Liquid Culture

Honda

Sample	Medium
サンプル名	培地

PCR Cloning

Murata

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw REDOX cloning primer 59/(µL)	Rv REDOX primer Ver.2 64/(µL)	DNA CCAP 1023/1 /(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

Predenature	Denature	Annealing	Extension
95°C	98°C	64°C	72°C	72°C	12°C
5min	20sec	15sec	50sec	5min	long time

Denature-Annealing-Extension 25cycle

Gel Extraction

Murata

Lane	Sample
1	1kb ladder
2	9/8 SAMmt PCR product (5µL)
3	-
4	9/8 SAMmt PCR product (10µL)
5	9/8 SAMmt PCR product (10µL)
6	-

PCR

Honda

2K KAPA HiFi HotStart ReadyMix/(µL)	Fw Primer SAMmt cloning primer/(µL)	Rv Primer SAMmt cloning primer/(µL)	9/9 AT (Gel Extraction product)/(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
98°C	98°C	60°C	72°C	72°C	4°C
5min	20sec	15sec	90sec	5min	long time

Denature-Annealing-Extension 25cycle

9/10

Electrophoresis

Murata

Lane	Sample
1	-
2	-
3	1kb ladder (5µL)
4	9/9 REDOX PCR product (2µL)
5	9/9 SAMmt PCR product (2µL)
6	1kb ladder (5µL)
7	-
8	-

Miniprep/DNA Purification

Murata

Sample	Concentration/(µg/mL)	260/280	260/230
T7-RBS	290	1.90	1.83

Cloning PCR

Murata

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw REDOX cloning primer 59/(µL)	Rv REDOX primer Ver.2 64/(µL)	DNA CCAP 1023/1 /(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

Electrophoresis

Murata

Lane	Sample
1	-
2	-
3	-
4	1kb ladder
5	SAMmt (x50) (10µL)
6	-
7	-
8	-

Colony PCR

Honda

Master Mix

Quick Tag/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
40	1.6	1.6	36.8	80

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	24sec

Denature-Annealing-Extension 30cycle

Number	Sample
1	pSB1C3-dddD(1)
2	pSB1C3-dddD(2)
3	pSB1C3-dddD(3)
4	pSB1C3-dddD(4)
5	pSB1C3-dddD(5)
6	pSB1C3-dddD(6)
7	pSB1C3-dddD (control)
8	control

Electrophoresis

Honda

Lane	Sample
1	-
2	-
3	-
4	1kb ladder
5	9/9 dddD+psb1c3 (1)
6	9/9 dddD+psb1c3 (2)
7	9/9 dddD+psb1c3 (3)
8	9/9 dddD+psb1c3 (4)
9	1kb ladder
10	9/9 dddD+psb1c3 (5)
11	9/9 dddD+psb1c3 (6)
12	9/9 dddD+psb1c3 (control)
13	control
14	1kb ladder
15	-
16	-
17	-

Electrophoresis

Yasuda

Lane	Sample
1	-
2	-
3	1kb ladder
4	REDOX PCR product
5	-

Overlap Extention(OE)-PCR

Murata

10x Buffer for KOD-Plus-Ver.2	2mM dNTPs	25mM MgSO4	Primer(10µM each) #1	Template DNA #2	KOD-Plus-(1U/µL)	MilliQ	Total
5µL	5µL	3µL	1.5µL	1µL	1µL	33µL	50µL

(1)
#1: AT OE-A, AT OE-B
#2: 9/8 AT (DNA Purification Product)(55µg/mL)
(2)
#1: AT OE-C, AT OE-D
#2: 9/8 AT (DNA Purification Product)(55µg/mL)
(3)
#1: AT OE-2C, AT OE-D
#2: 9/8 AT (DNA Purification Product)(55µg/mL)
(4)
#1: SAMmt OE-A, SAMmt OE-B
#2: 9/9 SAMmt (Gel Extraction Product)(55µg/mL)
(5)
#1: SAMmt OE-C, SAMmt OE-D
#2: 9/9 SAMmt (Gel Extraction Product)(55µg/mL)
(6)
#1: DECARB OE-A, DECARB OE-B<
#2: 9/4 DECARB Cloning-4 (55µg/mL)
(7)
#1: DECARB OE-C, DECARB OE-D
#2: 9/4 DECARB Cloning-4 (55µg/mL)
(8)
#1: DiDECARB OE-A, DiDECARB OE-B
#2: 9/9 DiDECARB (DNA Purification Product) (55µg/mL)
(9)
#1: DiDECARB OE-C, DiDECARB OE-D
#2: 9/9 DiDECARB (DNA Purification Product) (55µg/mL)
(10)
#1: AT OE-C, AT OE-2B
#2: 9/8 AT (DNA Purification Product)(55µg/mL)

Genome Cloning

Yasuda

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw REDOX cloning primer 59/(µL)	Rv REDOX primer Ver.2 64/(µL)	DNA CCAP 1023/1 /(µL)	MilliQ/(µL)	Total/(µL)
50	3	3	4	40	100

Number	AnnealingTemperature
1	65.0 °C
2	64.3 °C
3	63.0 °C
4	61.1 °C
5	58.8 °C
6	56.9 °C
7	55.7 °C
8	55.0 °C

Gel Extraction

Yasuda
(1)

Lane	Sample
1	1kb ladder
2	9/10 OE-PCR(1)
3	9/10 OE-PCR(1)
4	-
5	9/10 OE-PCR(2)
6	-
7	9/10 OE-PCR(2)
8	-
9	1kb ladder
10	9/10 OE-PCR(3)
11	9/10 OE-PCR(3)

(2)

Lane	Sample
1	1kb ladder
2	9/10 OE-PCR(4)
3	9/10 OE-PCR(4)
4	-
5	9/10 OE-PCR(5)
6	9/10 OE-PCR(5)
7	1kb ladder

(3)

Lane	Sample
1	-
2	1kb ladder
3	9/10 OE-PCR(6)
4	9/10 OE-PCR(6)

(4)

Lane	Sample
1	-
2	1kb ladder
3	9/10 OE-PCR(7)
4	9/10 OE-PCR(7)

(5)

Lane	Sample
1	-
2	1kb ladder
3	9/10 OE-PCR(8)
4	9/10 OE-PCR(8)

(6)

Lane	Sample
1	1kb ladder
2	-
3	9/10 OE-PCR(9)
4	9/10 OE-PCR(9)
5	9/10 OE-PCR(10)
6	9/10 OE-PCR(10)

Ligation

Tatsui and Murata

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
YC placIII (SmaI cut)	1	9/8 dddD(DNA purification product)	0.6	(2x)Buffer	5	T4 Ligase	1	2.4	10

Liquid Culture

Honda

Sample	Medium
9/9 dddD+psB1C3-(1)	@

Restriction Enzyme Digestion

Shimizu

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
9/9 T7-RBS	6.9	-	-	1	1	H	3	-	18.1	30
control	1	-	-	-	-	H	1	-	8	10

OE-PCR

Shimizu

10x Buffer for KOD-Plus-Ver.2	2mM dNTPs	25mM MgSO4	Primer(10µM each) #1	Template DNA #2	KOD-Plus-(1U/µL)	MilliQ	Total
5µL	5µL	3µL	2µL	0.5µL	1µL	31µL	50µL

(1)
#1: SAMmt OE-A, SAMmt OE-D
#2: 9/10 SAMmt(4)(5) (Gel Extraction Product)
(2)
#1: DECARB OE-A, DECARB OE-D
#2: 9/10 DECARB(6)(7) (Gel Extraction Product)
(3)
#1: DiDECARB OE-A, DiDECARB OE-D
#2: 9/10 DiDECARB(8)(9) (Gel Extraction Product)
(3')
#1: DiDECARB OE-A, DiDECARB OE-D
#2: 9/10 DiDECARB(3)(9) (Gel Extraction Product)

Predenature	Denature	Annealing	Extension	Last Extention	Cooling
98°C	98°C	55°C	72°C	72°C	12°C
5min	20sec	15sec	2min10sec	5min	long time

Denature-Annealing-Extension 25cycle

Ligation

Shimizu

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
YCplacIII	1	dddD	1	(2x)Buffer	5	T4 Ligase	1	2	10

9/11

Miniprep

Murata

Sample	Concentration/(µg/mL)	260/280	260/230
9/10 dddD (liquid culture product)	165	1.88	2.83

DNA Purification

Matsumoto

Sample	Concentration/(µg/mL)	260/280	260/230
9/10 REDOX(5)(6) (genome cloning product)	55	1.56	1.32

OE-PCR

Shimizu

10x Buffer for KOD-Plus-Ver.2	2mM dNTPs	25mM MgSO4	Primer(10µM each) #1	Template DNA #2	KOD-Plus-(1U/µL)	MilliQ	Total
5µL	5µL	3µL	1.5µL	1µL	1µL	32µL	50µL

(1)
#1: AT OE-A, AT OE-B
#2: 9/8 AT (DNA Purification Product)(55µg/mL)
(2)
#1: AT OE-C, AT OE-D
#2: 9/8 AT (DNA Purification Product)(55µg/mL)
(3)
#1: AT OE-2C, AT OE-D
#2: 9/8 AT (DNA Purification Product)(55µg/mL)
(4)
#1: REDOX (Forward with prefix), REDOX (Reverse with sufix) #2: 9/11 REDOX (DNA Purification Product)(55µg/mL) (5)(6)
#1: SAMmt OE-A, SAMmt OE-B
#2: 9/9 SAMmt (Gel Extraction Product)(55µg/mL)
(7)(8)
#1: SAMmt OE-C, SAMmt OE-D
#2: 9/9 SAMmt (Gel Extraction Product)(55µg/mL)
(9)(10)
#1: DECARB OE-A, DECARB OE-B<
#2: 9/4 DECARB Cloning-4 (55µg/mL)
(11)(12)
#1: DECARB OE-C, DECARB OE-D
#2: 9/4 DECARB Cloning-4 (55µg/mL)
(13)(14)
#1: DiDECARB OE-A, DiDECARB OE-B
#2: 9/9 DiDECARB (DNA Purification Product) (55µg/mL)
(15)(16)
#1: DiDECARB OE-C, DiDECARB OE-D
#2: 9/9 DiDECARB (DNA Purification Product) (55µg/mL)

Electrophoresis

Honda and Murata

Lane	Sample
1	-
2	1kb ladder
3	9/10 OE-PCR-(1)
4	9/10 OE-PCR-(2)
5	9/10 OE-PCR-(3)
6	9/10 OE-PCR-(3')
7	1kb ladder

Colony PCR

Murata and Yamauchi
Master Mix (x32)

Quick Taq/(µL)	M13 Fw/(µL)	M13 Rv/(µL)	MilliQ/(µL)	Total/(µL)
160	6.4	6.4	147.2	320

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	72sec

Denature-Annealing-Extension 30cycle

(1)

Number	Sample
1	LB Amp. 9/10 mamB(1)
2	LB Amp. 9/10 mamB(2)
3	LB Amp. 9/10 mamB(3)
4	LB Amp. 9/10 mamB(4)
5	LB Amp. 9/10 mamB(5)
6	LB Amp. 9/10 mamB(6)
7	LB Amp. 9/10 mamB(7)
8	LB Amp. 9/10 mamB(8)

(2)

Number	Sample
1	LB Amp. 9/10 mamL(1)
2	LB Amp. 9/10 mamL(2)
3	LB Amp. 9/10 mamL(3)
4	LB Amp. 9/10 mamL(4)
5	LB Amp. 9/10 mamL(5)
6	LB Amp. 9/10 mamL(6)
7	LB Amp. 9/10 mamL(7)
8	LB Amp. 9/10 mamL(8)

(3)

Number	Sample
1	LB Amp. 9/10 mamQ(1)
2	LB Amp. 9/10 mamQ(2)
3	LB Amp. 9/10 mamQ(3)
4	LB Amp. 9/10 mamQ(4)
5	LB Amp. 9/10 mamQ(5)
6	LB Amp. 9/10 mamQ(6)
7	LB Amp. 9/10 mamQ(7)
8	LB Amp. 9/10 mamQ(8)

(4)

Number	Sample
1	LB Amp. 9/10 dddD(1)
2	LB Amp. 9/10 dddD(2)
3	LB Amp. 9/10 dddD(3)
4	LB Amp. 9/10 dddD(4)
5	LB Amp. 9/10 dddD(5)
6	LB Amp. 9/10 dddD(6)
7	LB Amp. 9/10 dddD(7)
8	LB Amp. 9/10 dddD(8)

Mutation PCR

Honda
(1)dddD (PstI)

Prime STAR MAX Premix(2x)	Fw dddD PstI mutation primer	Rv dddD PstI mutation primer	9/11 MP dddD (x2000)	MilliQ	Total
25µL	1µL	1µL	1µL	22µL	50µL

(2)T7-RBS-Histag

Prime STAR MAX Premix(2x)	Fw psB1C3-his-F-v2 primer	Rv psB1C3 Histag mutation primer	9/10 MP T7-RBS (x3000)	MilliQ	Total
25µL	1µL	1µL	1µL	22µL	50µL

(3)Control

Prime STAR MAX Premix(2x)	Fw Control primer	Rv Control primer	Control template (x1000)	MilliQ	Total
25µL	0.5µL	0.5µL	1µL	23µL	50µL

Predenature	Denature	Annealing	Extension	Last Extention	Cooling
98°C	98°C	55°C	72°C	72°C	12°C
5min	10sec	15sec	25sec	5min	long time

Denature-Annealing-Extension 30cycle

DNA Purification

Honda

Sample	Concentration/(µg/mL)	260/280	260/230
dddD, T7-RBS-His	-	-	-

Gel Extraction

Honda
(1)

Lane	Sample
1	1kb ladder
2	9/11 OE-PCR-(1)
3	9/11 OE-PCR-(1)
4	-
5	-
6	9/11 OE-PCR-(2)
7	9/11 OE-PCR-(2)
8	-
9	1kb ladder
10	9/11 OE-PCR-(3)
11	9/11 OE-PCR-(3)

(2)

Lane	Sample
1	1kb ladder
2	9/11 OE-PCR-(6)
3	9/11 OE-PCR-(6)
4	-
5	-
6	9/11 OE-PCR-(8)
7	9/11 OE-PCR-(8)
8	-
9	1kb ladder
10	9/11 OE-PCR-(10)
11	9/11 OE-PCR-(10)

(3)

Lane	Sample
1	1kb ladder
2	9/11 OE-PCR-(12)
3	9/11 OE-PCR-(12)
4	9/11 OE-PCR (REDOX 2nd PCR)
5	-
6	9/11 OE-PCR-(14)
7	9/11 OE-PCR-(14)
8	-
9	1kb ladder
10	9/11 OE-PCR-(16)
11	9/11 OE-PCR-(16)

DNA Purification

Yamauchi

Sample	Concentration/(µg/mL)	260/280	260/230
9/10 OE-PCR (1) SAMmt	975	1.06	1.08
9/10 OE-PCR (2) DECARB	420	1.67	2.19
9/20 OE-PCR (3) DiDECARB	175	1.66	2.45
9/10 OE-PCR (3') DiDECARB	215	1.57	1.82
9/11 Mutation PCR dddD	275	1.61	1.92
9/11 Mutation PCR T7	140	1.49	1.78
Control C	340	1.62	1.47

Ligation

Tatsui and Yamauchi

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
YC placIII (SmaI cut)	1	9/11 SAMmt (DNA purification product)	0.5	(2x) Buufer	5	T4 Ligase	1	2.5	9

YC placIII (SmaI cut)19/11 DiDECARB (DNA purification product)1(2x) Buufer5T4 Ligase129 YC placIII (SmaI cut)19/11 DECARB (DNA purification product)1(2x) Buufer5T4 Ligase129 YC placIII (SmaI cut)19/11 REDOX (DNA purification product)2(2x) Buufer5T4 Ligase119 9/10 Pcon-X(PstI cut, gel extraction product)1.5mamL(X.P)2(2x)Buffer5T4 Ligase1-9.5 9/10 Pcon-X(PstI cut, gel extraction product)1.5mamQ(X.P)2(2x)Buffer5T4 Ligase1-9.5

OE-PCR

Yasuda

10x Buffer for KOD-Plus-Ver.2	2mM dNTPs	25mM MgSO4	Primer(10µM each) #1	Template DNA #2	KOD-Plus-(1U/µL)	MilliQ	Total
5µL	5µL	3µL	2µL	0.5µL	1µL	31µL	50µL

(1)
#1: AT OE-A, AT OE-D
#2: 9/10 OE-PCR(10), 9/11 OE-PCR(1), 9/11 OE-PCR(3)
(2)
#1: SAMmt OE-A, SAMmt OE-D
#2: 9/11 OE-PCR(6), 9/11 OE-PCR(8)
(3)
#1: DECARB OE-A, DECARB OE-D
#2: 9/11 OE-PCR(10), 9/11 OE-PCR(12)
(4)
#1: DiDECARB OE-A, DiDECARB OE-D
#2: 9/11 OE-PCR(14), 9/11 OE-PCR(16)

Predenature	Denature	Annealing	Extension	Last Extention	Cooling
98°C	98°C	55°C	72°C	72°C	12°C
2min	10sec	10sec	1min	5min	long time

Denature-Annealing-Extension 25cycle

9/12

Electrophoresis

Li

Lane	Sample
1	1kb ladder
2	9/11 OE-PCR 2nd (1)
3	9/11 OE-PCR 2nd (2)
4	9/11 OE-PCR 2nd (3)
5	9/11 OE-PCR 2nd (4)

DNA Purification

Li

Sample	Concentration/(µg/mL)	260/280	260/230
9/11 OE-PCR 2nd (1)	220	1.65	1.96
9/11 OE-PCR 2nd (2)	240	1.65	2.10
9/11 OE-PCR 2nd (3)	225	1.68	2.03
9/11 OE-PCR 2nd (4)	160	1.64	1.91

Transformation

Yamauchi

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
9/11 SAMmt (ligation product)	2	DH5&alfa;	10	12	SOD@
9/11 DiDECARB (ligation product)	2	DH5&alfa;	10	12	SOD
9/11 DECARB (ligation product)	2	DH5&alfa;	10	12	SOD
9/11 REDOX (ligation product)	2	DH5&alfa;	10	12	SOD

Sequence@

Yasuda

Bis Dye v3.1 Sequencing RR-24(/µL)	Bis Dye 5x Sequencing Buffer(/µL)	Primer (10µM) #1(/µL)	9/11 dddD miniprep (165ng/nL)(/µL)	MilliQ	Total
4	2	3.2	1.5	9.3	20

1. VF 2. Sequence No.1
3. Sequence No.2
4. Sequence No.3
5. Sequence No.4
6. Sequence No.5
7. Sequence No.6

Liquid Culture

Honda

Sample	Medium
9/10 dddD-YCplacIII (Transformation product)	SOD@

Restriction Enzyme Digestion

Murata

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
9/10 OE-PCR SAMmt	2	1	-	-	1	H	3	-	23	30
9/10 OE-PCR DECARB	4.8	-	1	1	-	M	3	3	17.2	30
9/10 OE-PCR DiDECARB	12.4	-	1	1	-	M	3	3	10.6	30
9/10 OE-PCR T7-RBS	6.9	-	-	1	1	H	3	-	18.1	30
Control	2.3	-	-	-	-	H	1	-	5	9.9

Genome Cloning PCR

Honda
(1)

2K KAPA HiFi HotStart ReadyMix/(µL)	Fw Primer SAMmt cloning primer/(µL)	Rv Primer SAMmt cloning primer/(µL)	DNA CCAP 1023/1 /(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

(2)

2K KAPA HiFi HotStart ReadyMix/(µL)	Fw Primer DECARB cloning primer/(µL)	Rv Primer DECARB cloning primer/(µL)	DNA CCAP 1023/1/(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
98°C	98°C	60°C	72°C	72°C	12°C
5min	20sec	15sec	90sec	5min	long time

Denature-Annealing-Extension 25cycle

Gel Extraction

Honda

Lane	Sample
1	1kb ladder
2	Control(9/11 T7-RBS)
3	-
4	9//11 T7-RBS(S,P)
5	-
6	1kb ladder

Cloning PCR (for Histag)

Murata

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw 9/10 AT OE-A/(µL)	Rv 9/12 AT His/(µL)	Templete 9/11 OE-PCR 2nd (1) 9/12/(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	65°C	72°C	72°C	12°C
5min	20sec	15sec	45sec	5min	long time

Denature-Annealing-Extension 25cycle

Miniprep

Tatsui

Sample	Concentration/(µg/mL)	260/280	260/230
dddD-psB1C3 (Liquid culture product)	370	1.75	2.48

DNA Purification

Tatsui

Sample	Concentration/(µg/mL)	260/280	260/230
9/11 DECARB (X,S)	-	-	-
9/11 DiDECARB (X,S)	-	-	-
9/11 SAMmt (X,S)	-	-	-

Restriction Enzyme Digestion

Tatsui

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
9/12 AT (DNA purification product)	9	1	-	-	1	H	3	-	16	30

PCR (for Control)

Murata
(1)

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw AT-A/(µL)	Rv AT-D/(µL)	Templete 9/8 AT (DNA purification product)/(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

(2)

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw SAMmt-A/(µL)	Rv SAMmt-D/(µL)	Templete 9/12 AT (Genome cloning product)/(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

(3)

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw DECARB-A/(µL)	Rv DECARB-D/(µL)	Templete 9/12 DECARB (genome cloning product)/(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

(4)

2x KAPA HiFi HotStart ReadyMix/(µL)	Fw DiDECARB-A/(µL)	Rv DiDECARB-D/(µL)	Templete 9/8 AT (DNA purification product)/(µL)	MilliQ/(µL)	Total/(µL)
12.5	0.75	0.75	1	10	25

Predenature	Denature	Annealing	Extension	Final Extension	Cooling
95°C	98°C	65°C	72°C	72°C	12°C
5min	20sec	15sec	45sec	5min	long time

Denature-Annealing-Extension 25cycle

Restriction Enzyme Digestion

Yamauchi

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
9/12 AT-His (DNA purification product)	8.7	-	1	-	1	M	3	3	13.3	30
Control	0.4	-	-	-	-	M	3	0	6.6	10
8/2 Pcon	20	1	-	1	-	H	3	3	2	30
Control	pick up	-	-	-	-	H	3	0	7	10

Ligation

Honda 111.5

Vector/(µL)	Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
YC placIII	AT-His cloning	0.5	(2x)Buffer	5	T4 Ligase	1	3.5	10
YC placIII	AT	0.5	(2x)Buffer	5	T4 Ligase	1	3.5	10
psB1C3 (E,P)	SAMmt (E,P)	1	(2x)Buffer	5	T4 Ligase	1	2.5	10

DNA Purification

Tatsui

Sample	Concentration/(µg/mL)	260/280	260/230
9/11 DECARB (Restriction Enzyme Digestion product)	40	1.74	1.95
9/11 DiDECARB (Restriction Enzyme Digestion product)	30	2.00	1.70
9/11 SAMmt (Restriction Enzyme Digestion product)	20	3.54	2.33
9/11 T7-RBS	60	1.81	0.27
9/10 T7-RBS	50	1.65	1.77

Electrophoresis

Lane	Sample
1	-
2	-
3	-
4	1kb ladder
5	9/11 E-PCR 2nd (1)
6	9/11 Control (PCR product) (1)
7	9/11 E-PCR 2nd (2)
8	9/11 Control (PCR product) (2)
9	9/11 E-PCR 2nd (3)
10	9/11 Control (PCR product) (3)
11	9/11 E-PCR 2nd (4)
12	9/11 Control (PCR product) (4)
13	1kb ladder
14	-
15	-
16	-

Transformation

Honda

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
AT-His-YCplacIII	1	DH5α	10	11	SOD
AT-YCplacIII	1	DH5α	10	11	SOD
SAMmt(E,P)-psB1C3(E,P)	1	DH5α	10	11	SOD

Transformation

Yasuda

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
9/11 dddD (DNA purification product)	1	DH5α	10	11	SOD
9/11 T7-His (DNA purification product)	1	DH5α	10	11	SOD

Colony PCR@

Tatsui and Li

Electrophoresis@

Tatsui and Li

Lane	Sample
1	YCplacIII SAMmt(1)
2	YCplacIII SAMmt(2)
3	YCplacIII SAMmt(3)
4	YCplacIII SAMmt(4)
5	YCplacIII SAMmt(5)
6	YCplacIII SAMmt(6)
7	YCplacIII SAMmt(7)
8	YCplacIII SAMmt(8)
9	1kb ladder
10	YCplacIII REDOX(1)
11	YCplacIII REDOX(2)
12	YCplacIII REDOX(3)
13	YCplacIII REDOX(4)
14	YCplacIII REDOX(5)
15	YCplacIII REDOX(6)
16	YCplacIII REDOX(7)

Liquid Culture@

Yamauchi AmpCPCP

Sample	Medium
9/5 Pcon-psB1C3-(3)	CP
9/5 Pcon-psB1C3-(3)	CP
9/11 SAMmt-(3)
9/8 T7-RBS-(1)
9/8 T7-RBS-(1)

Colony PCR

Honda

Master Mix

Quick Tag/(µL)	M13 Fw/(µL)	M13 Rv/(µL)	MilliQ/(µL)	Total/(µL)
35	1.4	1.4	32.2	70

1. REDOX-YCplacIII-(1)
2. REDOX-YCplacIII-(2)
3. REDOX-YCplacIII-(3)
4. REDOX-YCplacIII-(4)
5. REDOX-YCplacIII-(5)
6. REDOX-YCplacIII-(6)
7. REDOX-YCplacIII-(7)

Predenature	Denature	Annealing	Extension	Last Extention	Cooling
94°C	94°C	55°C	68°C	68°C	12°C
2min	30sec	30sec	1min	1sec	long time

Denature-Annealing-Extension 25cycle

Electrophoresis@

Honda

Lane	Sample
1
2
3
4
5
6
7
8
9	1kb ladder
10	REDOX-YCplacIII-(1)
11	REDOX-YCplacIII-(2)
12	REDOX-YCplacIII-(3)
13	REDOX-YCplacIII-(4)
14	REDOX-YCplacIII-(5)
15	REDOX-YCplacIII-(6)
16	REDOX-YCplacIII-(7)
17	REDOX-YCplacIII-(8)

9/13

Miniprep

Honda

Sample	Concentration/(µg/mL)	260/280	260/230
9/12 T7-RBS-psB1C3 (liquid culture product)	95	2.02	2.72
9/12 SAMmt-YCplacIII (liquid culture product)	110	1.70	1.15

Gel Extraction

Honda and Yasuda

Lane	Sample
1	1kb ladder
2	-
3	-
4	psB1C3-Pcon(X,S)
5	-
6	Control (psB1C3-Pcon(X,S))

Miniprep/DNA Purification

Yasuda

Sample	Concentration/(µg/mL)	260/280	260/230
9/12 AT (E,P)	2.3	1.33	1.11
9/12 AT-His(X,P)	2.0	1.33	0.86

Ligation

Honda

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
9/8 psB1C3(E,P)	3	9/13 AT(E,P)(DNA purification product)	1	(2x)Buffer	5	T4 Ligase	1	0	10
9/13 psB1C3(X,P)	3	9/13 AT-His(X,P)(DNA purification product)	1	(2x)Buffer	5	T4 Ligase	1	0	10

Miniprep

Honda

Sample	Concentration/(µg/mL)	260/280	260/230
9/12 SAMmt-YCplacIII	140	1.80	1.90
T7-RBS-psB1C3	290	1.78	1.65

Liquid Culture

Yasuda

Sample	Medium
9/12 dddD-psB1C3-(1)	CP
9/12 dddD-psB1C3-(2)	CP
9/12 dddD-psB1C3-(3)	CP
9/12 T7-RBS-His-(1)	CP
9/12 T7-RBS-His-(2)	CP
9/12 T7-RBS-His-(3)	CP
Control

9/14

Colony PCR

Yasuda

Quick Taq/(µL)	M13 Fw/(µL)	M13 Rv/(µL)	MilliQ/(µL)	Total/(µL)
335	13.4	13.4	368.2	670

Predenature	Denature	Annealing	Extension
94°C	94°C	55°C	68°C
2min	30sec	30sec	160sec

Denature-Annealing-Extension 30cycle

1~8: 9/12 AT-YCplacIII(1)~(8)
9~16: 9/12 AT His-YCplacIII(1)~(8)
17~24: 9/12 mamB-YCplacIII(1)~(8)
25~44: 9/12 DECARB-YCplacIII(1)~(20)
45~64: 9/12 DiDECARB-YCplacIII(1)~(20)

Gel Extraction

Honda

Lane	Sample
1	1kb ladder
2	9/13 Control (SAMmt)
3	9/13 SAMmt (E,P)
4	-
5	9/13 Control (Pcon-psB1C3)
6	9/13 Pcon-psB1C3(X,S)

DNA Purification

Honda

Sample	Concentration/(µg/mL)	260/280	260/230
Pcon-psB1C3(X,S)	40	0.13	1.27

Electrophoresis

Yasuda
(1)

Lane	Sample
1	9/12 AT-YCplacIII(1)
2	9/12 AT-YCplacIII(2)
3	9/12 AT-YCplacIII(3)
4	9/12 AT-YCplacIII(4)
5	9/12 AT-YCplacIII(5)
6	9/12 AT-YCplacIII(6)
7	9/12 AT-YCplacIII(7)
8	9/12 AT-YCplacIII(8)
9	1kbp GereRuler
10	9/12 AT His-YCplacIII(1)
11	9/12 AT His-YCplacIII(2)
12	9/12 AT His-YCplacIII(3)
13	9/12 AT His-YCplacIII(4)
14	9/12 AT His-YCplacIII(5)
15	9/12 AT His-YCplacIII(6)
16	9/12 AT His-YCplacIII(7)
17	9/12 AT His-YCplacIII(8)

(2)

Lane	Sample
1	9/12 mamB-YCplacIII(1)
2	9/12 mamB-YCplacIII(2)
3	9/12 mamB-YCplacIII(3)
4	9/12 mamB-YCplacIII(4)
5	9/12 mamB-YCplacIII(5)
6	9/12 mamB-YCplacIII(6)
7	9/12 mamB-YCplacIII(7)
8	9/12 mamB-YCplacIII(8)
9	1kbp GereRuler
10	9/12 DECARB-YCplacIII(1)
11	9/12 DECARB-YCplacIII(2)
12	9/12 DECARB-YCplacIII(3)
13	9/12 DECARB-YCplacIII(4)
14	9/12 DECARB-YCplacIII(5)
15	9/12 DECARB-YCplacIII(6)
16	9/12 DECARB-YCplacIII(7)
17	9/12 DECARB-YCplacIII(8)

(3)

Lane	Sample
1	9/12 DECARB-YCplacIII(9)
2	9/12 DECARB-YCplacIII(10)
3	9/12 DECARB-YCplacIII(11)
4	9/12 DECARB-YCplacIII(12)
5	9/12 DECARB-YCplacIII(13)
6	9/12 DECARB-YCplacIII(14)
7	9/12 DECARB-YCplacIII(15)
8	9/12 DECARB-YCplacIII(16)
9	1kbp GereRuler
10	9/12 DECARB-YCplacIII(17)
11	9/12 DECARB-YCplacIII(18)
12	9/12 DECARB-YCplacIII(19)
13	9/12 DECARB-YCplacIII(20)
14	9/12 DiDECARB-YCplacIII(1)
15	9/12 DiDECARB-YCplacIII(2)
16	9/12 DiDECARB-YCplacIII(3)
17	9/12 DiDECARB-YCplacIII(4)

(4)

Lane	Sample
1	9/12 DiDECARB-YCplacIII(5)
2	9/12 DiDECARB-YCplacIII(6)
3	9/12 DiDECARB-YCplacIII(7)
4	9/12 DiDECARB-YCplacIII(8)
5	9/12 DiDECARB-YCplacIII(9)
6	9/12 DiDECARB-YCplacIII(10)
7	9/12 DiDECARB-YCplacIII(11)
8	9/12 DiDECARB-YCplacIII(12)
9	1kbp GereRuler
10	9/12 DiDECARB-YCplacIII(13)
11	9/12 DiDECARB-YCplacIII(14)
12	9/12 DiDECARB-YCplacIII(15)
13	9/12 DiDECARB-YCplacIII(16)
14	9/12 DiDECARB-YCplacIII(17)
15	9/12 DiDECARB-YCplacIII(18)
16	9/12 DiDECARB-YCplacIII(19)
17	9/12 DiDECARB-YCplacIII(20)

Miniprep

Honda

Sample	Concentration/(µg/mL)	260/280	260/230
9/13 dddD-psB1C3(1)	645	1.82	1.70
9/13 dddD-psB1C3(2)	660	1,69	1.74
9/13 dddD-psB1C3(3)	635	1.87	1.57
9/13 T7-RBS-His-psB1C3(1)	440	1.65	1.23
9/13 T7-RBS-His-psB1C3(2)	450	1.92	1.46
9/13 T7-RBS-His-psB1C3(3)	190	1.44	1.26

Restriction Enzyme Digestion

Honda

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
9/14 dddD-psB1C3(1)	6	-	2	-	2	M	6	6	38	60
9/14 dddD-psB1C3(2)	6	-	2	-	2	M	6	6	38	60
9/14 dddD-psB1C3(3)	6	-	2	-	2	M	6	6	38	60
9/14 T7-RBS-His-psB1C3(1)	5	-	-	1	1	H	3	-	18	30
9/14 T7-RBS-His-psB1C3(2)	5	-	-	1	1	H	3	-	18	30
9/14 T7-RBS-His-psB1C3(3)	10	-	-	1	1	H	3	-	15	30

Dephosphorylation

Honda

Sample/(µL)		CIAP/(µL)
9/14 psB1C3(X,S)(gel extraction product)	20	0.25

Sample	Concentration/(µg/mL)	260/280	260/230
9/14 psB1C3(X,S)(gel extraction product)	60	2.93	0.16

Ligation

Yasuda

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
Pcon-psB1C3(X,S)(Dephosphorylation product)	0.83	9/12 DECARB(X,S)(DNA purification product)	2.93	10x Lig Buffer	2	NEB T4 DNA Ligase	1	13.24	20
Pcon-psB1C3(X,S)(Dephosphorylation product)	0.83	9/12 DECARB(X,S)(DNA purification product)	3.66	10x Lig Buffer	2	NEB T4 DNA Ligase	1	12.51	20