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アメフラシノート

7/31

Restriction Enzyme Digestion

Nakamura Matsumoto

Sample	Volume	EcoR1	Spe1	Buffer(H)	MilliQ	TOTAL
3/24 PCR(dddD)cloning	3.2	1	1	3	21.8	30
pSB1C3	18.5	1	1	3	6.5	30
pSB1C3(control)	6.2	0	0	0	2.8	10

8/1

Gel Extraction

Yasuda Murata

Lane	DNA
1	1kbp ladder
2	3/24 PCR(dddD)cloning E,S
3	3/24 PCR(dddD)cloning E,S
4	Blank
5	pSB1C3
6	pSB1C3
7	Blank
8	1kbp ladder
9	3/24 PCR(dddD)cloning(control)
10	pSB1C3(control)

8/1

Ligation

Yasuda

Vector	inserter	10xt4LigaseBuffer	t4Ligase	MilliQ	TOTAL
1.5 µL(8/1 pSB1C3 2047bp 13.3µg/mL 20μg)	6.9 µL(8/1 dddD 2545bp 10.8µg/mL 74.60µg)	2µL	1µL	8.6µL	20µL

8/1

transformation

Murata

Name	Sample/(µl)	Competent Cells/(µl)	Total/(µl)	Plate
pSB1C3	3	30	165	SOC(132µl)

8/2

PCR Cloning

Murata

	dddD 121µg/µL
2x KAPA HiFiHotStartReadyMix	12.5µL
F Pre-dddD cloning 62	0.75µL
Rv Spe1-dddDcloning 68	0.75µL
MilliQ	11µL
TOTAL	25µL

8/2

Colony PCR

Honda Matsumoto Nakamura

	Master Mix
Quick Taq	15µL
VF2	0.6µL
VR	0.6µL
MilliQ	13.8µL
TOTAL	30µL

8/2

Restriction Enzyme Digestion

Nakamura Matsumoto

Sample	Volume	EcoR1	Spe1	Buffer	MilliQ	TOTAL
8/2 dddD cloning	8.2	1	1	3	16.8	30
8/2 dddD cloning control	0.41	0	0	1	8.6	10
pSB1C3(control)	6.2	0	0	3(H)	2.8	10

8/3

Electrophoresis for confirmation

Murata

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

8/3

Gel Extraction

Tatsui

Lane	DNA
1	1kbp ladder
2	Blank
3	pSB1C3 25µμL 2070bp
4	Blank
5	pSB1C3 5µL
6	Blank
7	8/2 cloning dddD 25µL 2544bp
8	Blank
9	3/24 PCR(dddD)cloning(control)
10	8/2 cloning dddD 5µL

9/14

Gel Extraction

Honda,Yasuda

Lane	DNA
1	1kbp ladder
2	3/24 PCR(dddD)cloning E,S
3	9/13SAMmT(control)
4	9/13SAMmT(EP)
5	9/13Pcon-pSB1C3(control)
6	9/13Pcon-pSB1C3(XS)

9/13

Column cleanup

Yasuda

Sample	density(µg/mL)	260/280	260/230
9/12 AT-EP	2.3	1.33	1.11
9/12 AT-His-XP	2.0	1.33	0.86

9/13

Ligation

Honda

sumple	Vector	Insert	Ligase	2xBuffer	MilliQ
9/13 column cleanup AT(EP)+9/8 pSB1C3(EP)	1	3	1	5	0
9/13 column cleanup AT-His(XP)+9/13 pSB1C3(XP)	1	3	1	5	0

9/13

Miniprep

Honda

Sample	density(µg/mL)	260/280	260/230
9/12 SAMmt-YCPlac111	140	1.80	1.90
9/12 T7-RBS-psB1C3	290	1.78	1.65

9/13

Liquid Culture

Yasuda

Sample	Medium
9/12 dddD-pSB1C3	CP
9/12 dddD-pSB1C3	CP
9/12 dddD-pSB1C3	CP
9/12 T7-RBS-His	CP
9/12 T7-RBS-His	CP
9/12 T7-RBS-His	CP
control	CP

9/14

Colony PCR

Yasuda

	Master Mix
Quick Taq	335
VF	13.4
VR	13.4
MilliQ	308.2
TOTAL	670

9/14

Gel Extraction

Honda

Lane	DNA	volume
1	1kbp ladder	5
2	3/24 PCR(dddD)cloning E,S	15
3	9/13SAMmT(control)	15
4	9/13SAMmT(EP)	15
5	9/13Pcon-pSB1C3(control)	15
6	9/13Pcon-pSB1C3(XS)

9/14

Electrophoresis for confirmation

Honda, Li and Yamauchi

Lane	Restriction Enzyme Digestion Products
1	9/12 AT-YcPlac 1~8
2	9/12 AT-YcPlac 1~8
3	9/12 AT-YcPlac 1~8
4	9/12 AT-YcPlac 1~8
5	9/12 AT-YcPlac 1~8
6	9/12 AT-YcPlac 1~8
7	9/12 AT-YcPlac 1~8
8	9/12 AT-YcPlac 1~8
9	GeneRuler
10	9/12 AT-His-YcPlac 1~8
11	9/12 AT-His-YcPlac 1~8
12	9/12 AT-His-YcPlac 1~8
13	9/12 AT-His-YcPlac 1~8
14	9/12 AT-His-YcPlac 1~8
15	9/12 AT-His-YcPlac 1~8
16	9/12 AT-His-YcPlac 1~8
17	9/12 AT-His-YcPlac 1~8

Lane	Restriction Enzyme Digestion Products
1	9/12 mamB-YcPlac 1~8
2	9/12 mamB-YcPlac 1~8
3	9/12 mamB-YcPlac 1~8
4	9/12 mamB-YcPlac 1~8
5	9/12 mamB-YcPlac 1~8
6	9/12 mamB-YcPlac 1~8
7	9/12 mamB-YcPlac 1~8
8	9/12 mamB-YcPlac 1~8
9	GeneRuler
10	9/12 DECARB-YcPlac 1~8
11	9/12 DECARB-YcPlac 1~8
12	9/12 DECARB-YcPlac 1~8
13	9/12 DECARB-YcPlac 1~8
14	9/12 DECARB-YcPlac 1~8
15	9/12 DECARB-YcPlac 1~8
16	9/12 DECARB-YcPlac 1~8
17	9/12 DECARB-YcPlac 1~8

Lane	Restriction Enzyme Digestion Products
1	9/12 DECARB 9~16
2	9/12 DECARB 9~16
3	9/12 DECARB 9~16
4	9/12 DECARB 9~16
5	9/12 DECARB 9~16
6	9/12 DECARB 9~16
7	9/12 DECARB 9~16
8	9/12 DECARB 9~16
9	GeneRuler-YcPlac
10	9/12 DECARB-YcPlac 17~20
11	9/12 DECARB-Ycplac 17~20
12	9/12 DECARB-Ycplac 17~20
13	9/12 DiDECARB-YcPlac 1~4
14	9/12 DiDECARB-YcPlac 1~4
15	9/12 DiDECARB-YcPlac 1~4
16	9/12 DiDECARB-YcPlac 1~4
17	9/12 DiDECARB-YcPlac 1~4

Lane	Restriction Enzyme Digestion Products
1	9/12 DiDECARB-YcPlac 5~12
2	9/12 DiDECARB-YcPlac 5~12
3	9/12 DiDECARB-YcPlac 5~12
4	9/12 DiDECARB-YcPlac 5~12
5	9/12 DiDECARB-YcPlac 5~12
6	9/12 DiDECARB-YcPlac 5~12
7	9/12 DiDECARB-YcPlac 5~12
8	9/12 DiDECARB-YcPlac 5~12
9	GeneRuler
10	9/12 DiDECARB-YcPlac 13~20
11	9/12 DiDECARB-YcPlac 13~20
12	9/12 DiDECARB-YcPlac 13~20
13	9/12 DiDECARB-YcPlac 13~20
14	9/12 DiDECARB-YcPlac 13~20
15	9/12 DiDECARB-YcPlac 13~20
16	9/12 DiDECARB-YcPlac 13~20
17	9/12 DiDECARB-YcPlac 13~20

9/14

Miniprep

Honda

DNA	density(µg/ml)	260/280	260/230
9/14 dddD(µ)-pSB1C3	645	1.82	1.70
9/14 dddD(µ)-pSB1C3	660	1.69	1.74
9/14 dddD(µ)-pSB1C3	635	1.87	1.57
9/14 T7-RBS-His-pSB1C3	440	1.65	1.23
9/14 T7-RBS-His-pSB1C3	450	1.92	1.46
9/14 T7-RBS-His-pSB1C3	190	1.ss	1.26

9/14

Restriction Enzyme Digestion

Honda

Sample	Volume	Spe1	Pst1	Xba1)	Buffur	BSA	MilliQ	TOTAL
9/14 dddD(µ)-pSB1C3	6	0	2	2	6(M)	6	38	60
9/14 dddD(µ)-pSB1C3	6	0	2	2	6(M)	6	38	60
9/14 dddD(µ)-pSB1C3	6	0	2	2	6(M)	6	38	60
9/14 T7-RBS-His-pSB1C3	5	1	1	0	3(H)	0	18	30
9/14 T7-RBS-His-pSB1C3	5	1	1	0	3(H)	0	18	30
9/14 T7-RBS-His-pSB1C3	10	1	1	0	3(H)	0	15	30

9/14

Ligation

Yasuda

NEB T4DNALigase	10XLig Buffer	9/12 DECARB XS column	9/14 Pcon-pSB1C3 XS Dephosphorylated	MilliQ	TOTAL
1	2	2.93	0.83	13.24	20

NEB T4DNALigase	10XLig Buffer	9/12 DiDECARB XS column	9/14 Pcon-pSB1C3 XS Dephosphorylated	MilliQ	TOTAL
1	2	3.66	0.83	12.5	20