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LabNote

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DesignNote

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	CuMTSE62	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	CuMTSE62	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 12th.

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	CuMTSE62	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/Marker		Insert	Enzyme	Buffer
Lane	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 14th and June 11th.

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		induced
BL21(DE3)pLysS	pGEX6P-2	CuMTSE1	induced
BL21(DE3)pLysS	pGEX6P-2	CuMTS2	induced
BL21(DE3)pLysS	pGEX6P-2		non-induced
BL21(DE3)pLysS	pGEX6P-2	CuMTS1	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	CuMTS2	induced
BL21(DE3)pLysS	pGEX6P-2		non-induced
BL21(DE3)pLysS	pGEX6P-2	CuMTS1	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	CuMTS2	induced
BL21(DE3)pLysS	pGEX6P-2		non-induced
BL21(DE3)pLysS	pGEX6P-2	CuMTS1	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	CuMTSE11
BL21(DE3)pLysS	pGEX6P-2	CuMTSE2

PCR

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

AGE

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

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	P-F1,P-R
CuMTSE2	P-F2,P-R

Restriction Digest

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

DNA Refinement2

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

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

Transformation

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

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	P-F1,P-R	1～4
DH5α	p427-TEF	CuMTSE1	P-F2,P-R	a～h
DH5α	p427-TEF	CuMTSE1	P-F2,P-R	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/Marker		Primer	Enzyme
Lane	Host	Insert	Primer	Enzyme
1	ikb Ladder
2		CuMTSE1	P-F1,P-R
3	4			Spe1,Xho1
4	4			Spe1,Xho1
5	4			N.D
6		CuMTSE1	P-F2,P-R
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	P-F1,P-R
BY4247	p427-TEF	CuMTSE1	P-F2,P-R

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	P-F1,P-R	A～H
BY4247	p427-TEF	CuMTSE1	P-F2,P-R	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)
Divide each of 2 plates into 8 sections and inoculate following samples on them.

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

July 2, 2014

ONISHI
Miniprep

Host	Sample
Host	Vector	Insert	Primer
BY4247	p426-TEF
BY4247	p427-TEF	CuMTSE1	P-F1,P-R
BY4247	p427-TEF	CuMTSE1	P-F2,P-R

Restriction Digest

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

AGE

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

July 3, 2014

OKAMOTO
PCR

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

Protein Extraction (Saccharomyces cerevisiae)

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

July 4, 2014

SHIMADA
AGE

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

PCR

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

AGE

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

July 5, 2014

KOBAYASHI
SDS-PAGE

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

July 6, 2014

SOGA
DNA Refinement1

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

Restriction Digest

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

DNA Refinement2

Vector	Sample		Enzyme
Vector	DNA	Primer	Enzyme
	CuMTSE2	P-F1,P-R	Spe1,Xho1
	CuMTSE2	P-F2,P-R	Spe1,Xho1

7/25

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August

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September

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October

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June

June 10, 2014

June 11, 2014

June 12, 2014

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June 14, 2014

June 16, 2014

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June 21, 2014

June 24, 2014

June 25, 2014

June 26, 2014

June 27, 2014

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July 1, 2014

July 2, 2014

July 3, 2014

July 4, 2014

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