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Protocol

LabNote

June

June 10, 2014

OKAMOTO
Miniprep

Host	Sample
Host	Vector	Insert
BL21(DE3)	pGEX6P-2	CuMTSE1
BL21(DE3)	pGEX6P-2	CuMTSE2
BL21(DE3)	pGEX6P-2	CuMTS3
BL21(DE3)	pGEX6P-2	CuMTS62

Restriction Digest

Host	Sample		Enzyme	Buffer
Host	Vector	Insert	Enzyme	Buffer
BL21(DE3)PlysS	pGEX6P-2	CuMTSE1	EcoR1	Buffer H
BL21(DE3)PlysS	pGEX6P-2	CuMTSE2	EcoR1	Buffer H
BL21(DE3)PlysS	pGEX6P-2	CuMTS3	EcoR1	Buffer H
BL21(DE3)PlysS	pGEX6P-2	CuMTS62	EcoR1	Buffer H
BL21(DE3)PlysS	pGEX6P-2	CuMTSE1	Sal1, Not1	Buffer H
BL21(DE3)PlysS	pGEX6P-2	CuMTSE2	Sal1, Not1	Buffer H
BL21(DE3)PlysS	pGEX6P-2	CuMTS3	Sal1, Not1	Buffer H
BL21(DE3)PlysS	pGEX6P-2	CuMTS62	Sal1, Not1	Buffer H

June 11, 2014

KOBAYASHI
AGE

Lane	Sample/Marker		Enzyme
Lane	Vector	Insert	Enzyme
1	λDNA-HindⅢ
2	pGEX6P-2	CuMTSE1	N.D
3	pGEX6P-2	CuMTSE1	EcoR1
4	pGEX6P-2	CuMTSE1	Sal1, Not1
5	pGEX6P-2	CuMTSE2	N.D
6	pGEX6P-2	CuMTSE2	EcoR1
7	pGEX6P-2	CuMTSE2	Sal1, Not1
8	pGEX6P-2	CuMTS3	N.D
9	pGEX6P-2	CuMTS3	EcoR1
10	pGEX6P-2	CuMTS62	N.D
11	pGEX6P-2	CuMTS62	EcoR1
12	pGEX6P-2	CuMTS62	Sal1, Not1

June 12, 2014

KOBAYASHI
Transformation

Host	Sample
Host	Vector	Insert
BL21(DE3)pLysS	pGEX6P-2	CuMTSE1
BL21(DE3)pLysS	pGEX6P-2	CuMTSE2
BL21(DE3)pLysS	pGEX6P-2	CuMTS3
BL21(DE3)pLysS	pGEX6P-2	CuMTS62

June 13, 2014

KOBAYASHI
Colony Isolation
Select 6 colonies per plate from 4 plates from June 12.

Host	Sample		Section
Host	Vector	Insert	Section
BL21(DE3)pLysS	pGEX6P-2	CuMTSE1	1-a, 1-b
BL21(DE3)pLysS	pGEX6P-2	CuMTSE2	2-a, 2-b
BL21(DE3)pLysS	pGEX6P-2	CuMTS3	3-a, 3-b
BL21(DE3)pLysS	pGEX6P-2	CuMTS62	6-a, 6-b

June 14, 2014

ONISHI
Miniprep

Host	Sample
Host	Vector	Insert
1-a	pGEX6P-2	CuMTSE1
1-b	pGEX6P-2	CuMTSE1
2-a	pGEX6P-2	CuMTSE2
2-b	pGEX6P-2	CuMTSE2
3-a	pGEX6P-2	CuMTS3
3-b	pGEX6P-2	CuMTS3
6-a	pGEX6P-2	CuMTS62
6-b	pGEX6P-2	CuMTS62

Miniprep

Host	Sample
Host	Vector	Insert
BL21(DE3)	pGEX6P-2	CuMTSE1
BL21(DE3)	pGEX6P-2	CuMTSE2
BL21(DE3)	pGEX6P-2	CuMTS3
BL21(DE3)	pGEX6P-2	CuMTS62

Restriction Digest

Host	Sample		Enzyme	Buffer
Host	Vector	Insert	Enzyme	Buffer
1-a	pGEX6P-2	CuMTSE1	EcoR1	Buffer H
1-b	pGEX6P-2	CuMTSE1	EcoR1	Buffer H
2-a	pGEX6P-2	CuMTSE2	EcoR1	Buffer H
2-b	pGEX6P-2	CuMTSE2	EcoR1	Buffer H
3-a	pGEX6P-2	CuMTS3	EcoR1	Buffer H
3-b	pGEX6P-2	CuMTS3	EcoR1	Buffer H
6-a	pGEX6P-2	CuMTS62	EcoR1	Buffer H
6-b	pGEX6P-2	CuMTS62	EcoR1	Buffer H
BL21(DE3)	pGEX6P-2	CuMTSE1	EcoR1	Buffer H
BL21(DE3)	pGEX6P-2	CuMTSE2	EcoR1	Buffer H
BL21(DE3)	pGEX6P-2	CuMTS3	EcoR1	Buffer H
BL21(DE3)	pGEX6P-2	CuMTS62	EcoR1	Buffer H

AGE

Lane	Sample			Insert	Enzyme	Buffer
Lane	Marker	Host	Vector	Insert	Enzyme	Buffer
1	1kb Ladder
2	λDNA-HindⅢ
3		1-a	pGEX6P-2	CuMTSE1	EcoR1	Buffer H
4		1-b	pGEX6P-2	CuMTSE1	EcoR1	Buffer H
5		2-a	pGEX6P-2	CuMTSE2	EcoR1	Buffer H
6		2-b	pGEX6P-2	CuMTSE2	EcoR1	Buffer H
7		3-a	pGEX6P-2	CuMTS3	EcoR1	Buffer H
8		3-b	pGEX6P-2	CuMTS3	EcoR1	Buffer H
9		6-a	pGEX6P-2	CuMTS62	EcoR1	Buffer H
10		6-b	pGEX6P-2	CuMTS62	EcoR1	Buffer H
11		BL21(DE3)	pGEX6P-2	CuMTSE1	EcoR1	Buffer H
12		BL21(DE3)	pGEX6P-2	CuMTSE2	EcoR1	Buffer H
13		BL21(DE3)	pGEX6P-2	CuMTS3	EcoR1	Buffer H
14		BL21(DE3)	pGEX6P-2	CuMTS62	EcoR1	Buffer H

Applied λDNA-HindⅢ to compare and contrast AGE of July 14 and June 11.

June 16, 2014

YAMAJI
Preculture

Host	Sample
Host	Vector	Insert
BL21(DE3)pLysS	pGEX6P-2	CuMTSE1
BL21(DE3)pLysS	pGEX6P-2	CuMTSE2
BL21(DE3)pLysS	pGEX6P-2	CuMTS3
BL21(DE3)pLysS	pGEX6P-2	CuMTS62
BL21(DE3)pLysS	pGEX6P-2

Main Culture

Host	Sample		IPTG
Host	Vector	Insert	IPTG
BL21(DE3)pLysS	pGEX6P-2	CuMTSE1	Induced
BL21(DE3)pLysS	pGEX6P-2	CuMTSE2	Induced
BL21(DE3)pLysS	pGEX6P-2	CuMTS3	Induced
BL21(DE3)pLysS	pGEX6P-2	CuMTS62	Induced
BL21(DE3)pLysS	pGEX6P-2		Induced
BL21(DE3)pLysS	pGEX6P-2	CuMTSE1	Non-induced
BL21(DE3)pLysS	pGEX6P-2	CuMTSE2	Non-induced
BL21(DE3)pLysS	pGEX6P-2	CuMTS3	Non-induced
BL21(DE3)pLysS	pGEX6P-2	CuMTS62	Non-induced
BL21(DE3)pLysS	pGEX6P-2		Non-induced

June 18, 2014

YAMAJI
Preculture

Host	Sample
Host	Vector	Insert
BL21(DE3)pLysS	pGEX6P-2	CuMTSE1
BL21(DE3)pLysS	pGEX6P-2	CuMTSE2

Main Culture

Host	Sample		IPTG
Host	Vector	Insert	IPTG
BL21(DE3)pLysS	pGEX6P-2		Induced
BL21(DE3)pLysS	pGEX6P-2	CuMTSE1	Induced
BL21(DE3)pLysS	pGEX6P-2	CuMTSE2	Induced
BL21(DE3)pLysS	pGEX6P-2		Non-induced
BL21(DE3)pLysS	pGEX6P-2	CuMTSE1	Non-induced
BL21(DE3)pLysS	pGEX6P-2	CuMTSE2	Non-induced

Protein Extraction (E.coli)

Host	Sample		IPTG
Host	Vector	Insert	IPTG
BL21(DE3)pLysS	pGEX6P-2		Induced
BL21(DE3)pLysS	pGEX6P-2	CuMTSE1	Induced
BL21(DE3)pLysS	pGEX6P-2	CuMTSE2	Induced
BL21(DE3)pLysS	pGEX6P-2		Non-induced
BL21(DE3)pLysS	pGEX6P-2	CuMTSE1	Non-induced
BL21(DE3)pLysS	pGEX6P-2	CuMTSE2	Non-induced

Preparations for SDS-PAGE
Add SDS sample buffer to protein extractions in a flask, incubate at 37℃ for 15 minutes and keep it at 4℃.

June 19, 2014

KITOMI
SDS-PAGE

Host	Sample		IPTG
Host	Vector	Insert	IPTG
BL21(DE3)pLysS	pGEX6P-2		Induced
BL21(DE3)pLysS	pGEX6P-2	CuMTSE1	Induced
BL21(DE3)pLysS	pGEX6P-2	CuMTSE2	Induced
BL21(DE3)pLysS	pGEX6P-2		Non-induced
BL21(DE3)pLysS	pGEX6P-2	CuMTSE1	Non-induced
BL21(DE3)pLysS	pGEX6P-2	CuMTSE2	Non-induced

June 21, 2014

SOGA
Miniprep

Host	Sample
Host	Vector	Insert
BL21(DE3)pLysS	pGEX6P-2	CuMTSE1
BL21(DE3)pLysS	pGEX6P-2	CuMTSE2

PCR

Vector	Sample		DNA Polymerase
Vector	Insert	Primer	DNA Polymerase
pGEX6P-2	CuMTSE1	pGEX6P-2F1, pGEX6P-2R	KOD-FX-NEO
pGEX6P-2	CuMTSE2	pGEX6P-2F2, pGEX6P-2R	KOD-FX-NEO
pGEX6P-2	CuMTSE1	pGEX6P-2F1, pGEX6P-2R	KOD-FX-NEO
pGEX6P-2	CuMTSE2	pGEX6P-2F2, pGEX6P-2R	KOD-FX-NEO

AGE

Lane	Sample			Insert	Primer
Lane	Marker	Host	Vector	Insert	Primer
1	1kb Ladder
2		BY4247	p427-TEF
3			pGEX6P-2	CuMTSE1	pGEX6P-2F1, pGEX6P-2R
4			pGEX6P-2	CuMTSE2	pGEX6P-2F2, pGEX6P-2R
5			pGEX6P-2	CuMTSE1	pGEX6P-2F1, pGEX6P-2R
6			pGEX6P-2	CuMTSE2	pGEX6P-2F2, pGEX6P-2R

June 24, 2014

SOGA
Preculture

Host	Sample
Host	Vector	Insert
BL21(DE3)pLysS
BL21(DE3)pLysS	pGEX6P-2	CuMTSE1
BL21(DE3)pLysS	pGEX6P-2	CuMTSE2

June 25, 2014

KOBAYASHI

Cultivated following samples in Big Scale at 30℃ overnight in shaken culture.

Host	Sample
Host	Vector	Insert
BL21(DE3)pLysS
BL21(DE3)pLysS	pGEX6P-2	CuMTSE1
BL21(DE3)pLysS	pGEX6P-2	CuMTSE2

DNA Refinement1

Sample
DNA	Primer
CuMTSE1	pGEX6P-2F1, pGEX6P-2R
CuMTSE2	pGEX6P-2F2, pGEX6P-2R

Restriction Digest

Vector	Sample		Enzyme	Buffer
Vector	DNA	Primer	Enzyme	Buffer
	CuMTSE1	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1	Buffer H
	CuMTSE2	pGEX6P-2F2, pGEX6P-2R	Spe1, Xho1	Buffer H
p427-TEF			Spe1, Xho1	Buffer H

DNA Refinement2

Vector	Sample		Enzyme
Vector	DNA	Primer	Enzyme
	CuMTSE1	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1
	CuMTSE2	pGEX6P-2F2, pGEX6P-2R	Spe1, Xho1
p427-TEF			Spe1, Xho1

Main Culture

Host	Sample		IPTG
Host	Vector	Insert	IPTG
BL21(DE3)pLysS	pGEX6P-2		Induced
BL21(DE3)pLysS	pGEX6P-2	CuMTSE1	Induced
BL21(DE3)pLysS	pGEX6P-2	CuMTSE2	Induced
BL21(DE3)pLysS	pGEX6P-2		Non-induced
BL21(DE3)pLysS	pGEX6P-2	CuMTSE1	Non-induced
BL21(DE3)pLysS	pGEX6P-2	CuMTSE2	Non-induced

Ligation

Sample
Vector	DNA	Primer
p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R
p427-TEF	CuMTSE1	pGEX6P-2F2, pGEX6P-2R
p427-TEF	CuMTSE2	pGEX6P-2F1, pGEX6P-2R
p427-TEF	CuMTSE2	pGEX6P-2F2, pGEX6P-2R

DNA ligase: DNA Ligation Kit ‹Mighty Mix› (TAKARA BIO INC.)

Transformation

Host	Sample
Host	Vector	Insert	Primer
DH5α	p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R
DH5α	p427-TEF	CuMTSE1	pGEX6P-2F2, pGEX6P-2R
DH5α	p427-TEF	CuMTSE2	pGEX6P-2F1, pGEX6P-2R
DH5α	p427-TEF	CuMTSE2	pGEX6P-2F2, pGEX6P-2R

June 26, 2014

SAWANO
Colony Isolation
Isolate each colony from the plates which were inoculated on June 25.

Host	Sample			Section
Host	Vector	Insert	Primer	Section
DH5α	p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R	1～4
DH5α	p427-TEF	CuMTSE1	pGEX6P-2F2, pGEX6P-2R	a～h
DH5α	p427-TEF	CuMTSE1	pGEX6P-2F2, pGEX6P-2R	ア～タ

June 27, 2014

SHIMADA
Colony Sweep

Lane	Sample
Lane	Host	Vector	Insert
1	BL21(DE3)pLysS	pGEX6P-2	CuMTSE1
2	1
3	2
4	3
5	4
6	A
7	B
8	C
9	D
10	E
11	F
12	G
13	H

Colony Sweep

Lane	Sample
Lane	Host	Vector	Insert
1	BL21(DE3)pLysS	pGEX6P-2	CuMTSE1
2	ア
3	イ
4	ウ
5	エ
6	オ
7	カ
8	キ
9	ク
10	ケ
11	コ
12	サ
13	シ
14	ス
15	セ
16	ソ
17	タ

Identified targeted genes in 4, c, h, イ, カ, セ

Miniprep

Sample
Host
4
C
H
イ
カ
セ

Restriction Digest

Sample	Enzyme	Buffer
Host	Enzyme	Buffer
4	Spe1, Xho1	Buffer H
C	Spe1, Xho1	Buffer H
H	Spe1, Xho1	Buffer H
イ	Spe1, Xho1	Buffer H
カ	Spe1, Xho1	Buffer H

AGE

Lane	Sample			Primer	Enzyme
Lane	Marker	Host	Insert	Primer	Enzyme
1	1kb Ladder
2			CuMTSE1	pGEX6P-2F1, pGEX6P-2R
3		4			Spe1, Xho1
4		4			Spe1, Xho1
5		4			N.D
6			CuMTSE1	pGEX6P-2F2, pGEX6P-2R
7		c			Spe1, Xho1
8		c			Spe1, Xho1
9		c			N.D
10		h			Spe1, Xho1
11		h			Spe1, Xho1
12		h			N.D

Transformation (electroportation)

Host	Sample
Host	Vector	Insert	Primer
BY4247	p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R
BY4247	p427-TEF	CuMTSE1	pGEX6P-2F2, pGEX6P-2R

June 30, 2014

SOGA
Inoculation
Divide each of 2 plates into 8 sections and inoculate following samples on them.

Host	Sample			Section
Host	Vector	Insert	Primer	Section
BY4247	p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R	A～H
BY4247	p427-TEF	CuMTSE1	pGEX6P-2F2, pGEX6P-2R	一～ハ

Cultivate 17 samples in a small scale at 30℃ for 6 hours in shaken culture.

July

July 1, 2014

KOBAYASHI
Protein Extraction (Saccharomyces cerevisiae)

Host	Sample
Host	Vector	Insert	Primer
BY4247	p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R
BY4247	p427-TEF	CuMTSE1	pGEX6P-2F2, pGEX6P-2R

July 2, 2014

ONISHI
Miniprep

Host	Sample
Host	Vector	Insert	Primer
BY4247	p427-TEF
BY4247	p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R
BY4247	p427-TEF	CuMTSE1	pGEX6P-2F2, pGEX6P-2R

Restriction Digest

Sample			Enzyme	Buffer
Vector	Insert	Primer	Enzyme	Buffer
p427-TEF	CuMTSE1		EcoR1	Buffer H
p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R	EcoR1	Buffer H
p427-TEF	CuMTSE1	pGEX6P-2F2, pGEX6P-2R	EcoR1	Buffer H
p427-TEF	CuMTSE1		Sac1, Kpn1	Buffer L
p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R	Sac1, Kpn1	Buffer L
p427-TEF	CuMTSE1	pGEX6P-2F2,pGEX6P-2R	Sac1, Kpn1	Buffer L

AGE

Lane	Sample				Enzyme
Lane	Marker	Vector	Insert	Primer	Enzyme
1		p427-TEF			N.D
2		p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R	N.D
3		p427-TEF	CuMTSE1	pGEX6P-2F2, pGEX6P-2R	N.D
4	1kb Ladder
5		p427-TEF			EcoR1
6		p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R	EcoR1
7		p427-TEF	CuMTSE1	pGEX6P-2F2, pGEX6P-2R	EcoR1
8	1kb Ladder
9		p427-TEF			Kpn1
10		p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R	Kpn1
11		p427-TEF	CuMTSE1	pGEX6P-2F2, pGEX6P-2R	Kpn1
12	1kb Ladder
13		p427-TEF			Sac1
14		p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R	Sac1
15		p427-TEF	CuMTSE1	pGEX6P-2F2, pGEX6P-2R	Sac1

Restriction Digest

Sample			Enzyme	Buffer
Vector	Insert	Primer	Enzyme	Buffer
p427-TEF	CuMTSE1		EcoR1	Buffer H
p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R	EcoR1	Buffer H
p427-TEF	CuMTSE1	pGEX6P-2F2, pGEX6P-2R	EcoR1	Buffer H
p427-TEF	CuMTSE1		Sac1	Buffer L
p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R	Sac1	Buffer L
p427-TEF	CuMTSE1	pGEX6P-2F2,pGEX6P-2R	Sac1	Buffer L
p427-TEF	CuMTSE1		Kpn1	Buffer L
p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R	Kpn1	Buffer L
p427-TEF	CuMTSE1	pGEX6P-2F2, pGEX6P-2R	Kpn1	Buffer L
p427-TEF	CuMTSE1		Spe1, Xho1	Buffer H
p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1	Buffer H
p427-TEF	CuMTSE1	pGEX6P-2F2, pGEX6P-2R	Spe1, Xho1	Buffer H

AGE

Lane	Sample				Enzyme
Lane	Marker	Vector	Insert	Primer	Enzyme
1	1kb Ladder
2		p427-TEF	CuMTSE1		EcoR1
3		p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R	EcoR1
4		p427-TEF	CuMTSE1	pGEX6P-2F2, pGEX6P-2R	EcoR1
5		p427-TEF	CuMTSE1		Kpn1
6		p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R	Kpn1
7		p427-TEF	CuMTSE1	pGEX6P-2F2, pGEX6P-2R	Kpn1
8		p427-TEF	CuMTSE1		Sac1
9		p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R	Sac1
10		p427-TEF	CuMTSE1	pGEX6P-2F2, pGEX6P-2R	Sac1
11		p427-TEF	CuMTSE1		Spe1, Xho1
12		p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1
13		p427-TEF	CuMTSE1	pGEX6P-2F2, pGEX6P-2R	Spe1, Xho1

July 3, 2014

OKAMOTO
PCR

Sample			DNA Polymerase
Vector	Insert	Primer	DNA Polymerase
pGEX6P-2	CuMTSE2	pGEX6P-2F1, pGEX6P-2R	KOD-FX-NEO
pGEX6P-2	CuMTSE2	pGEX6P-2F2, pGEX6P-2R	KOD-FX-NEO
pGEX6P-2	CuMTS3	pGEX6P-2F1, pGEX6P-2R	KOD-FX-NEO
pGEX6P-2	CuMTS3	pGEX6P-2F2, pGEX6P-2R	KOD-FX-NEO
pGEX6P-2	CuMTS62	pGEX6P-2F1, pGEX6P-2R	KOD-FX-NEO
pGEX6P-2	CuMTS62	pGEX6P-2F2, pGEX6P-2R	KOD-FX-NEO

Protein Extraction (Saccharomyces cerevisiae)

Host	Sample
Host	Vector	Insert	Primer
BY4247	p427-TEF
六	p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R
A	p427-TEF	CuMTSE1	pGEX6P-2F2, pGEX6P-2R

July 4, 2014

SHIMADA
AGE

Lane	Sample/Marker
Lane	Marker	Vector	Insert	Primer
1	1kb Ladder
2		pGEX6P-2	CuMTSE2	pGEX6P-2F1, pGEX6P-2R
3		pGEX6P-2	1kb Ladder+K83:N91	pGEX6P-2F2, pGEX6P-2R
4		pGEX6P-2	CuMTS3	pGEX6P-2F1, pGEX6P-2R
5		pGEX6P-2	CuMTS3	pGEX6P-2F2, pGEX6P-2R
6		pGEX6P-2	CuMTS62	pGEX6P-2F1, pGEX6P-2R
7		pGEX6P-2	CuMTS62	pGEX6P-2F2, pGEX6P-2R

PCR

Sample			DNA Polymerase
Vector	Insert	Primer	DNA Polymerase
pGEX6P-2	CuMTSE2	pGEX6P-2F1, pGEX6P-2R	KOD-FX-NEO
pGEX6P-2	CuMTSE2	pGEX6P-2F2, pGEX6P-2R	KOD-FX-NEO
pGEX6P-2	CuMTS3	pGEX6P-2F1, pGEX6P-2R	KOD-FX-NEO
pGEX6P-2	CuMTS3	pGEX6P-2F2, pGEX6P-2R	KOD-FX-NEO
pGEX6P-2	CuMTS62	pGEX6P-2F1, pGEX6P-2R	KOD-FX-NEO
pGEX6P-2	CuMTS62	pGEX6P-2F2, pGEX6P-2R	KOD-FX-NEO

AGE

Lane	Sample
Lane	Marker	Vector	Insert	Primer
1	1kb Ladder
2		pGEX6P-2	CuMTSE2	pGEX6P-2F1, pGEX6P-2R
3		pGEX6P-2	1kb Ladder+K83:N91	pGEX6P-2F2, pGEX6P-2R
4		pGEX6P-2	CuMTS3	pGEX6P-2F1, pGEX6P-2R
5		pGEX6P-2	CuMTS3	pGEX6P-2F2, pGEX6P-2R
6		pGEX6P-2	CuMTS62	pGEX6P-2F1, pGEX6P-2R
7		pGEX6P-2	CuMTS62	pGEX6P-2F2, pGEX6P-2R

July 5, 2014

KOBAYASHI
SDS-PAGE

Host	Sample
Host	Vector	Insert	Primer
BL21(DE3)pLysS	pGEX6P-2
4	pGEX6P-2	CuMTSE1	pGEX6P-2F1, pGEX6P-2R
C	pGEX6P-2	CuMTSE2	pGEX6P-2F2, pGEX6P-2R
BY4247	p427-TEF
六	p427-TEF	CuMTSE1	pGEX6P-2F1, pGEX6P-2R
A	p427-TEF	CuMTSE2	pGEX6P-2F2, pGEX6P-2R

July 6, 2014

SOGA
DNA Refinement1

Sample
DNA	Primer
CuMTSE1	pGEX6P-2F1, pGEX6P-2R
CuMTSE2	pGEX6P-2F2, pGEX6P-2R
CuMTS3	pGEX6P-2F1, pGEX6P-2R
CuMTS62	pGEX6P-2F1, pGEX6P-2R

Restriction Digest

Vector	Sample		Enzyme	Buffer
Vector	DNA	Primer	Enzyme	Buffer
	CuMTSE2	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1	Fast digest buffer
	CuMTSE2	pGEX6P-2F2, pGEX6P-2R	Spe1, Xho1	Fast digest buffer
	CuMTS3	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1	Fast digest buffer
	CuMTS62	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1	Fast digest buffer
p427-TEF			Spe1, Xho1	Fast digest buffer

DNA Refinement2

Sample		Enzyme
DNA	Primer	Enzyme
CuMTSE2	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1
CuMTSE2	pGEX6P-2F2, pGEX6P-2R	Spe1, Xho1

Ligation

Vector	Sample		Enzyme
Vector	DNA	Primer	Enzyme
p427-TEF	CuMTSE2	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1
p427-TEF	CuMTSE2	pGEX6P-2F2, pGEX6P-2R	Spe1, Xho1

DNA ligase: DNA Ligation Kit ‹Mighty Mix› (TAKARA BIO INC.)

Transformation (E.coli)

Host	Sample			Enzyme
Host	Vector	Insert	Primer	Enzyme
DH5α	p427-TEF	CuMTSE2	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1
DH5α	p427-TEF	CuMTSE2	pGEX6P-2F2, pGEX6P-2R	Spe1, Xho1
DH5α	p427-TEF	CuMTS3	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1
DH5α	p427-TEF	CuMTS62	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1

July 7, 2014

KOBAYASHI
Colony Isolation
Isolate each colony from the plates which were inoculated on July 6.

Host	Sample			Section
Host	Vector	Insert	Primer	Section
DH5α	p427-TEF	CuMTSE2	pGEX6P-2F1, pGEX6P-2R	1～16
DH5α	p427-TEF	CuMTS3	pGEX6P-2F1, pGEX6P-2R	ア～エ
DH5α	p427-TEF	CuMTS62	pGEX6P-2F1, pGEX6P-2R	a～p

Transformation (E.coli)

Host	Sample			Enzyme
Host	Vector	Insert	Primer	Enzyme
DH5α	p427-TEF	CuMTSE2	pGEX6P-2F2, pGEX6P-2R	Spe1, Xho1

July 8, 2014

OKAMOTO
Colony Sweep

Lane	Sample
Lane	Host
1	DH5α	p427-TEF
2	a
3	b
4	c
5	d
6	e
7	f
8	g
9	h
10	i
11	j
12	k
13	l
14	m
15	n
16	o
17	p

Colony Sweep

Lane	Sample
Lane	Host
1	DH5α	p427-TEF
2	ア
3	イ
4	ウ
5	エ

Colony Sweep

Lane	Sample
Lane	Host
1	DH5α	p427-TEF
2	1
3	2
4	3
5	4
6	5
7	6
8	7
9	8
10	9
11	10
12	11
13	12
14	13
15	14
16	15
17	16

July 9, 2014

OKAMOTO
Main Culture

Sample
Host
d
h
l
ア
イ
エ

Mini Prep

Sample
Host
D
H
L
ア
イ
エ

Restriction Digest

Vector	Sample		Enzyme	Buffer
Vector	Insert	Primer	Enzyme	Buffer
p427-TEF(d)	CuMTS3	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1	Fast digest buffer
p427-TEF(h)	CuMTS3	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1	Fast digest buffer
p427-TEF(l)	CuMTS3	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1	Fast digest buffer
p427-TEF(ア)	CuMTS62	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1	Fast digest buffer
p427-TEF(イ)	CuMTS62	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1	Fast digest buffer
p427-TEF(エ)	CuMTS62	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1	Fast digest buffer

AGE

Lane	Sample				Enzyme
Lane	Marker	Vector	Insert	Primer	Enzyme
1	1kb Ladder
2		p427-TEF(ア)	CuMTS3	pGEX6P-2F1, pGEX6P-2R	N.D
3		p427-TEF(ア)	CuMTS3	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1
4		p427-TEF(イ)	CuMTS3	pGEX6P-2F1, pGEX6P-2R	N.D
5		p427-TEF(イ)	CuMTS3	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1
6		p427-TEF(エ)	CuMTS3	pGEX6P-2F1, pGEX6P-2R	N.D
7		p427-TEF(エ)	CuMTS3	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1
8		p427-TEF(d)	CuMTS62	pGEX6P-2F1, pGEX6P-2R	N.D
9		p427-TEF(d)	CuMTS62	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1
10		p427-TEF(h)	CuMTS62	pGEX6P-2F1, pGEX6P-2R	N.D
11		p427-TEF(h)	CuMTS62	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1
12		p427-TEF(l)	CuMTS62	pGEX6P-2F1, pGEX6P-2R	N.D
13		p427-TEF(l)	CuMTS62	pGEX6P-2F1, pGEX6P-2R	Spe1, Xho1

Marker: 1kb Ladder

August

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From 2014.igem.org

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