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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/td>
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/td>
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

July

7/25

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August

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September

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October

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From 2014.igem.org

LabNote

June

June 10, 2014

June 11, 2014

June 12, 2014

June 13, 2014

June 14, 2014

June 16, 2014

June 19, 2014

June 21, 2014

June 24, 2014

June 25, 2014

June 26, 2014

June 27, 2014

July

7/25

August

September

October