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	コンピ名	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 Tag/(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

@

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	@	1.37	0.42
dddD PCR product	35	1.54	0.85

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°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 Tag/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
5	0.2	0.2	4.6	10

(2)

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

(3)

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

(4)

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

control(2)

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

(4)

Quick Tag/(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 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	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	@
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	@	50	61.2	SOC
8/19 Ligation (2)	11.2	@	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	@	20	25	SOC
control	5	@	20	25	SOC

@

8/21

Colony PCR

Honda,Nakamura

Master Mix

Quick Tag/(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
@	@

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	@	50	60	SOC
8/25 Ligation(2)	10	@	50	60	SOC
8/25 Ligation(3)	10	@	30	40	SOC

@

8/27

Colony PCR

Murata

Master Mix

Quick Tag/(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

Quick Tag/(µL)	VF2/(µL)	VR/(µL)	MilliQ/(µL)	Total/(µL)
@	@	@	@	25

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

Denature-Annealing-Extension @cycle

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	@	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	-	3	-	17.2	30
I732095 8/22 miniprep (2)(255ng/µL)	0.4	-	-	-	-	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)
I732095 8/22 miniprep (2)(255ng/µL)	7.8	1	-	1	-	3	-	17.2	30
I732095 8/22 miniprep (2)(255ng/µL)	0.4	-	-	-	-	1	-	8.6	10
I732095 8/22 miniprep (1)(430ng/µL)	4.6	1	-	1	-	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

ゲノムクローニング@

Honda

Electrophoresis

Honda (1)AT

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

<
(2)SAMmt

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

<
(3)DiDECARB

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

9/8

ゲノムクローニング@

Honda and Yamaura

PCR@

Honda and Yamaura

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

カラム精製

Matsumoto

Restriction Enzyme Digestion

Matsumoto

Sample/(µL)		EcoR1/(µL)	Xba1/(µL)	Spe1/(µL)	Pst1/(µL)	Buffer/(µL)		BSA/(µL)	MilliQ/(µL)	Total/(µL)
サンプル名	(サンプル)	(E)	(X)	(S)	(P)	(HorM)	(Buffer)	(BSA)	(MilliQ)	30
control	(サンプル)	(E)	(X)	(S)	(P)	(HorM)	(Buffer)	(BSA)	(MilliQ)	10

PCR

Yasuda

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	9	T4 Ligase	1	-	18
pSB1C3	4.2	-	-	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@

Murata

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, AT 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

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