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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 PCR(dddD) cloning product	3.2	1	-	1	-	H	3	-	21.8	30
7/18 RBS30@	18.5	1	-	1	-	H	3	-	6.5	30
7/18 RBS30 control@	6.2	-	-	-	-	H	1	-	2.8	10

8/1

Gel Extraction

Murata and Yasuda

Lane	Sample
1	1kbp ladder	2	3/24 PCR(dddD) cloning product E,S	3	3/24 PCR(dddD) cloning product E,S	4	Blank	5	7/18 RBS30 E,S@	6	7/18 RBS30 E,S@	7	Blank	8	1kbp ladder	9	3/24 PCR(dddD) cloning product control	10	7/18 RBS30 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	コンピ名	30	33	SOC

8/2

PCR Cloning

Murata

@	2x KAPA HiFi Hotstart Ready Mix/(µL)	F Pre-dddD cloning 62/(µL)	Rv Spe1-dddD cloning 68/(µL)	MilliQ/(µL)	Total/(µL)
@	12.5	0.75	0.75	11	25

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

Denature-Annealing-Extension 25cycle

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

DNA Purification

Murata

@

Colony PCR

Honda,Nakamura and Matsumoto

Master Mix

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

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

Denature-Annealing-Extension 30cycle

☆

Concentration Measurement

Nakamura and Matsumoto

Sample	Concentration/(µg/mL)	260/280	260/230
@	245	1.59	2.17

Restriction Enzyme Digestion

名前

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
8/2 dddD cloning product@	8.2	1	-	1	-	H	3	-	16.8	30
8/2 dddD cloning control@	0.41	-	-	-	-	H	1	-	8.6	10
7/18 RBS30@	12.7	1	-	1	-	H	3	-	12.3	30
7/18 RBS30 control@	0.04	-	-	-	-	H	1	-	8.4	10

8/3

@

Murata

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

@

Tatsui

Lane	Sample
1	1kb ladder	2	Blank	3	7/18 RBA30@(pSB1C3) 2070bp	4	Blank	5	7/18 RBA30@(pSB1C3)	6	Blank	7	8/2 cloning dddD 2544bp	8	Blank	9	8/2 cloning dddD

Sample	Concentration/(µg/mL)	260/280	260/230
7/18 RBS30(pSB1C3)@	@	1.37	0.42
8/2 cloning dddD	35	1.54	0.85

Ligation

@

Vector/(µL)		Insert/(µL)		Buffer/(µL)		Ligase/(µL)		MilliQ/(µL)	Total/(µL)
@	7	@	7	2xBuffer	15	T4Ligase	1	@	@
@	7	@	14	2xBuffer	22	T4Ligase	1	@	@
@	7	@	@	2xBuffer	15	T4Ligase	1	7	@

Transformation

@

Sample/(µL)		CompetentCells/(µL)		Total/(µL)	Medium
@	@	@	@	@	@

8/4

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@	94@	55@	68@
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

@

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