Team:KIT-Kyoto/Notebook/Labnote

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LabNote

June

June 10, 2014

OKAMOTO
Miniprep

Host Sample
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
Vector Insert
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 CuMTS62 Sal1, Not1 Buffer H


June 11, 2014

KOBAYASHI
AGE

Lane Sample/Marker Enzyme
Vector Insert
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
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
Vector Insert
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
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
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
Vector Insert
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
Host Vector
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
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
Vector Insert
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
Vector Insert
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
Vector Insert
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
Vector Insert
BL21(DE3)pLysS pGEX6P-2 CuMTSE1
BL21(DE3)pLysS pGEX6P-2 CuMTSE2

PCR
Vector Sample DNA Polymerase
Insert Primer
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
Host Vector
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
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
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
DNA Primer
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
DNA Primer
CuMTSE1 P-F1, P-R Spe1, Xho1
CuMTSE2 P-F2, P-R Spe1, Xho1
p427-TEF Spe1, Xho1

Main Culture
Host Sample IPTG
Vector Insert
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
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
Vector Insert Primer
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
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
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
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
Host Insert
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
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
Vector Insert Primer
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
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
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
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
Vector Insert Primer
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
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
Host 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
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
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
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
DNA Primer
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
Sample Enzyme
DNA Primer
CuMTSE2 P-F1, P-R Spe1, Xho1
CuMTSE2 P-F2, P-R Spe1, Xho1

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

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

Transformation (E.coli)
Host Sample Enzyme
Vector Insert Primer
DH5α p427-TEF CuMTSE2 P-F1, P-R Spe1, Xho1
DH5α p427-TEF CuMTSE2 P-F2, P-R Spe1, Xho1
DH5α p427-TEF CuMTS3 P-F1, P-R Spe1, Xho1
DH5α p427-TEF CuMTS62 P-F1, P-R Spe1, Xho1



July 7, 2014

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

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

Transformation (E.coli)
Host Sample Enzyme
Vector Insert Primer
DH5α p427-TEF CuMTSE2 P-F2, P-R Spe1, Xho1


July 8, 2014

OKAMOTO
Colony Sweep

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

Colony Sweep
Lane Sample
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
Insert Primer
p427-TEF(d) CuMTS3 P-F1, P-R Spe1, Xho1 Fast digest buffer
p427-TEF(h) CuMTS3 P-F1, P-R Spe1, Xho1 Fast digest buffer
p427-TEF(l) CuMTS3 P-F1, P-R Spe1, Xho1 Fast digest buffer
p427-TEF(ア) CuMTS62 P-F1, P-R Spe1, Xho1 Fast digest buffer
p427-TEF(イ) CuMTS62 P-F1, P-R Spe1, Xho1 Fast digest buffer
p427-TEF(エ) CuMTS62 P-F1, P-R Spe1, Xho1 Fast digest buffer

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

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