Team:KIT-Kyoto/Notebook/Labnote

From 2014.igem.org

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<td>六</td>
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<td>p427-TEF</td>
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<td>CuMTSE2</td>
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<td>CuMTSE1</td>
<td>pGEX6P-2F2, pGEX6P-2R</td>
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(TOYOBO, KOD-Plus-Mutagenesis Kit)
(TOYOBO, KOD-Plus-Mutagenesis Kit)
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Start from protocol No.2.
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Start from KOD -Plus- Mutagenesis Kit protocol No.2.
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KOBAYASHI
KOBAYASHI
<br>
<br>
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Restart mutagenesis from protocol No.3 to obtain plasmid (muta-CuMTS3 in pGEX-6P-2)
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Restart mutagenesis from KOD -Plus- Mutagenesis Kit protocol No.3 to obtain plasmid (muta-CuMTS3 in pGEX-6P-2)
<br>
<br>
<strong>Transformation</strong>
<strong>Transformation</strong>
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<div class="accordion">
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KOBAYASHI
KOBAYASHI
<br>
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<strong>Main culture</strong>
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<strong>Main Culture</strong>
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KOBAYASHI
KOBAYASHI
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<strong>GC-MS analysis</strong>
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<strong>GC Analysis</strong>
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<div class="m_table">
<div class="m_table">

Latest revision as of 01:13, 18 October 2014

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) 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
BL21(DE3) pGEX6P-2 CuMTSE1 Sal1, Not1 Buffer H
BL21(DE3) pGEX6P-2 CuMTSE2 Sal1, Not1 Buffer H
BL21(DE3) pGEX6P-2 CuMTS3 Sal1, Not1 Buffer H
BL21(DE3) pGEX6P-2 CuMTS62 Sal1, Not1 Buffer H


June 11, 2014

KOBAYASHI
AGE

Lane Sample Enzyme
Marker Vector Insert
1 λDNA-HindⅢ
2 pGEX6P-2 CuMTSE1 Non Treated
3 pGEX6P-2 CuMTSE1 EcoR1
4 pGEX6P-2 CuMTSE1 Sal1, Not1
5 pGEX6P-2 CuMTSE2 Non Treated
6 pGEX6P-2 CuMTSE2 EcoR1
7 pGEX6P-2 CuMTSE2 Sal1, Not1
8 pGEX6P-2 CuMTS3 Non Treated
9 pGEX6P-2 CuMTS3 EcoR1
10 pGEX6P-2 CuMTS3 Sal1, Not1
11 pGEX6P-2 CuMTS62 Non Treated
12 pGEX6P-2 CuMTS62 EcoR1
13 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 Insert Enzyme Buffer
Marker 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

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

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 pGEX6P-2F1, pGEX6P-2R KOD-FX-NEO
pGEX6P-2 CuMTSE1 pGEX6P-2F2, pGEX6P-2R KOD-FX-NEO
pGEX6P-2 CuMTSE2 pGEX6P-2F1, pGEX6P-2R KOD-FX-NEO
pGEX6P-2 CuMTSE2 pGEX6P-2F2, pGEX6P-2R KOD-FX-NEO

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


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

Cultivate 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 Refinement 1
Sample
DNA Primer
CuMTSE1 pGEX6P-2F1, pGEX6P-2R
CuMTSE1 pGEX6P-2F2, pGEX6P-2R

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

DNA Refinement 2
Vector Sample Enzyme
DNA Primer
CuMTSE1 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1
CuMTSE1 pGEX6P-2F2, pGEX6P-2R 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 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
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
Vector Insert Primer
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
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 Primer Enzyme
Marker Host Insert
1 1kb Ladder
2 CuMTSE1 pGEX6P-2F1, pGEX6P-2R
3 4 Spe1, Xho1
4 4 Spe1, Xho1
5 4 Non Treated
6 CuMTSE1 pGEX6P-2F2, pGEX6P-2R
7 c Spe1, Xho1
8 c Spe1, Xho1
9 c Non Treated
10 h Spe1, Xho1
11 h Spe1, Xho1
12 h Non Treated

Transformation (electroportation)
Host Sample
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
Vector Insert Primer
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 (S. cerevisiae)

Host Sample
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
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
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
Marker Vector Insert Primer
1 p427-TEF Non Treated
2 p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R Non Treated
3 p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R Non Treated
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
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
Marker Vector Insert Primer
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
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 (S. cerevisiae)
Host Sample
Vector Insert Primer
BY4247 p427-TEF
A p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R
六 p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R


July 4, 2014

SHIMADA
AGE

Lane Sample/Marker
Marker Vector Insert Primer
1 1kb Ladder
2 pGEX6P-2 CuMTSE2 pGEX6P-2F1, pGEX6P-2R
3 pGEX6P-2 CuMTSE2 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
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
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
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
A p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R
六 p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R


July 6, 2014

SOGA
DNA Refinement 1

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

Ligation
Vector Sample Enzyme
DNA Primer
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
Vector Insert Primer
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
Vector Insert Primer
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
Vector Insert Primer
DH5α p427-TEF CuMTSE2 pGEX6P-2F2, pGEX6P-2R 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 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
Vector Insert Primer
1 p427-TEF(ア) CuMTS3 pGEX6P-2F1, pGEX6P-2R Non Treated
2 p427-TEF(ア) CuMTS3 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1
3 p427-TEF(イ) CuMTS3 pGEX6P-2F1, pGEX6P-2R Non Treated
4 p427-TEF(イ) CuMTS3 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1
5 p427-TEF(エ) CuMTS3 pGEX6P-2F1, pGEX6P-2R Non Treated
6 p427-TEF(エ) CuMTS3 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1
7 p427-TEF(d) CuMTS62 pGEX6P-2F1, pGEX6P-2R Non Treated
8 p427-TEF(d) CuMTS62 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1
9 p427-TEF(h) CuMTS62 pGEX6P-2F1, pGEX6P-2R Non Treated
10 p427-TEF(h) CuMTS62 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1
11 p427-TEF(l) CuMTS62 pGEX6P-2F1, pGEX6P-2R Non Treated
12 p427-TEF(l) CuMTS62 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1
Marker: 1kb Ladder

August

August 19, 2014

KOBAYASHI
Conduct mutagenesis to remove an illegal restriction site (EcoR1)
InversePCR

Sample
Vector Insert Primer DNA polymerase
pGEX6P-2 CuMTS3 Terpinene3-mutagenesis-FP, Terpinene3-mutagenesis-RP KOD-Plus
(TOYOBO, KOD-Plus-Mutagenesis Kit) Start from KOD -Plus- Mutagenesis Kit protocol No.2.

August 20, 2014

KOBAYASHI
Restart mutagenesis from KOD -Plus- Mutagenesis Kit protocol No.3 to obtain plasmid (muta-CuMTS3 in pGEX-6P-2)
Transformation

Sample
Host Vector Insert Primer
DH5α pGEX6P-2 muta-CuMTS3 Terpinene3-mutagenesis-FP and Terpinene3-mutagenesis-RP


August 21, 2014

KOBAYASHI
Colony Isolation
Isolate each colony from two plates which were inoculated overnight

Sample Section
Host Vector Insert Primer
DH5α pSB1C3 muta-CuMTS3 Terpinene3-mutagenesis-FP,
Terpinene3-mutagenesis-RP		 あ-み
Select 5 isolated colonies whose code names are あ,か,さ,た and な and miniprep them.

August 22, 2014

KOBAYASHI
Restriction Digest

Sample Enzyme Buffer
Host Vector Insert
DH5α あ pGEX6P-2 muta-CuMTS3 Sal1 Fast Digest buffer
DH5α か pGEX6P-2 muta-CuMTS3 Sal1 Fast Digest buffer
DH5α さ pGEX6P-2 muta-CuMTS3 Sal1 Fast Digest buffer
DH5α た pGEX6P-2 muta-CuMTS3 Sal1 Fast Digest buffer

AGE
Lane Sample Enzyme
Marker Host Vector Insert
1 1kb Ladder
2 DH5α あ pGEX6P-2 muta-CuMTS3 Sal1
3 DH5α か pGEX6P-2 muta-CuMTS3 Sal1
4 DH5α さ pGEX6P-2 muta-CuMTS3 Sal1
5 DH5α た pGEX6P-2 muta-CuMTS3 Sal1

Restriction Digest
Sample Enzyme Buffer
Host Vector Insert
DH5α か pGEX6P-2 CuMTS3 EcoR1 Fast Digest buffer
DH5α か pGEX6P-2 muta-CuMTS3 EcoR1 Fast Digest buffer

AGE
Lane Sample Enzyme
Marker Host Vector Insert
1 1kb Ladder
2 DH5α か pGEX6P-2 CuMTS3 EcoR1
3 DH5α か pGEX6P-2 muta-CuMTS3 EcoR1
An illegal EcoR1 restriction site was removed.

PCR
Sample DNA polymerase
Vector Insert Primer
か pGEX6P-2 muta-CuMTS3 terpinene3-prefix and terpinene3-suffix KOD-FX-NEO-Plus

AGE
Lane Sample DNA polymerase
Marker Vector Insert Primer
1 1kb Ladder
2 か pGEX6P-2 muta-CuMTS3 terpinene3-prefix and terpinene3-suffix KOD-FX-NEO-Plus


August 30, 2014

KOBAYASHI
PCR
Sample DNA polymerase
Vector Insert Primer
か pGEX6P-2 muta-CuMTS3 terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1 Taq

AGE
Lane Sample
Marker Vector Insert Primer
1 1kb Ladder
2 か pGEX6P-2 muta-CuMTS3 terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1

Made total amount of 295 μL of PCR samples.

September

September 18, 2014

KOBAYASHI
Miniprep
Sample
Host Vector Insert
DH5α pSB1C3 BBa_J04450
DH5α pSB1C3 BBa_J04450
DH5α pSB1C3 BBa_J04450

Restriction Digest
Sample Buffer
Vector Insert Enzyme
pSB1C3 BBa_J04450 EcoR1, Pst1 Buffer H
pSB1C3 BBa_J04450 Xba1, Spe1 Fast Digest buffer
pSB1C3 BBa_J04450

AGE
Lane Sample Enzyme
Marker Host Vector Insert
1 1kb Ladder
2 DH5α pSB1C3 BBa_J04450 EcoR1, Pst1
3 DH5α pSB1C3 BBa_J04450 Xba1, Spe1
4 DH5α pSB1C3 BBa_J04450

PCR
Sample DNA polymerase
Vector Insert Primer
か pGEX6P-2 muta-CuMTS3 terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1 Taq

DNA Refinement 1
Sample
Insert Primer
muta-CuMTS3 terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1

Restriction Digest
Sample Enzyme Buffer
Vector Insert Primer
pSB1C3 Xba1, Spe1 Fast Digest
か pGEX6P-2 muta-CuMTS3 terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1 Xba1, Spe1 Fast Digest

DNA Refinement 2
Sample
Insert Primer
muta-CuMTS3 terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1

Ligation
Sample Enzyme
Vector DNA Primer
pSB1C3 muta-CuMTS3 terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1 Spe1, Xba1
DNA ligase: DNA Ligation Kit ‹Mighty Mix› (TAKARA BIO INC.)

Transformation
Sample
Host Vector Insert Primer
DH5α pSB1C3 muta-CuMTS3 terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1

September 19, 2014

KOBAYASHI
Colony Isolation
Isolate each colony from the plates which were inoculated overnight.
Sample Section
Host Vector Insert Primer
DH5α pSB1C3 muta-CuMTS3 terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1 A-P
DH5α pSB1C3 muta-CuMTS3 terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1 ア-タ


Colony Sweep
Lane Sample
Host Vector Insert
1 DH5α pSB1C3 muta-CuMTS3
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

Targeted colonies were identified in セ and ソ.

Colony Sweep
Lane Sample
Host Vector Insert
1 DH5α pSB1C3 muta-CuMTS3
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


Targeted colonies were identified in colony N and F.

Select セ, ソ, N, F

Main Culture
Sample
Host Vector Insert Primer
DH5α(N) pSB1C3 muta-CuMTS3 ----------
DH5α(F) pSB1C3 muta-CuMTS3 ----------
DH5α(セ) pSB1C3 muta-CuMTS3 ----------
DH5α(ソ) pSB1C3 muta-CuMTS3 ----------

Cultivate these samples in 3mL of LB medium with ChlPhe at 30°C overnight.

September 20, 2014

KOBAYASHI
Miniprep
Sample
Host Vector Primer
DH5α(N) pSB1C3 muta-CuMTS3
DH5α(F) pSB1C3 muta-CuMTS3
DH5α(セ) pSB1C3 muta-CuMTS3
DH5α(ソ) pSB1C3 muta-CuMTS3

Enzyme: E/P and Xba1

Restriction Digest
Sample Enzyme Buffer
Marker Host Vector Insert
pSB1C3
DH5α (セ) pSB1C3 muta-CuMTS3
DH5α (ソ) pSB1C3 muta-CuMTS3
DH5α (F) pSB1C3 muta-CuMTS3
DH5α (N) pSB1C3 muta-CuMTS3
DH5α (セ) pSB1C3 EcoR1, Pst1 Buffer H
DH5α (ソ) pSB1C3 muta-CuMTS3 EcoR1, Pst1 Buffer H
DH5α (F) pSB1C3 muta-CuMTS3 EcoR1, Pst1 Buffer H
DH5α (N) pSB1C3 muta-CuMTS3 EcoR1, Pst1 Buffer H
pSB1C3 muta-CuMTS3 EcoR1, Pst1 Buffer H
pSB1C3 Xba1 FD buffer
DH5α (セ) pSB1C3 muta-CuMTS3 Xba1 FD buffer
DH5α (ソ) pSB1C3 muta-CuMTS3 Xba1 FD buffer
DH5α (F) pSB1C3 muta-CuMTS3 Xba1 FD buffer
DH5α (N) pSB1C3 muta-CuMTS3 Xba1 FD buffer

AGE
Lane Sample Enzyme
Marker Host Vector Insert
1 pSB1C3
2 DH5α (セ) pSB1C3 muta-CuMTS3
3 DH5α (ソ) pSB1C3 muta-CuMTS3
4 DH5α (F) pSB1C3 muta-CuMTS3
5 DH5α (N) pSB1C3 muta-CuMTS3
6 1kb Ladder
7 DH5α (セ) pSB1C3 muta-CuMTS3 EcoR1, Pst1
8 DH5α (ソ) pSB1C3 muta-CuMTS3 EcoR1, Pst1
9 DH5α (F) pSB1C3 muta-CuMTS3 EcoR1, Pst1
10 DH5α (N) pSB1C3 muta-CuMTS3 EcoR1, Pst1
11 pSB1C3 muta-CuMTS3 EcoR1, Pst1
12 1kb Ladder
13 pSB1C3 Xba1
14 DH5α (セ) pSB1C3 muta-CuMTS3 Xba1
15 DH5α (ソ) pSB1C3 muta-CuMTS3 Xba1
16 DH5α (F) pSB1C3 muta-CuMTS3 Xba1
17 DH5α (N) pSB1C3 muta-CuMTS3 Xba1

Sample F was selected as a submission plasmid.

Main Culture
Sample
Host Vector Insert Primer
DH5α(F) pSB1C3 muta-CuMTS3 ----------
DH5α(F) pSB1C3 muta-CuMTS3 ----------
DH5α(F) pSB1C3 muta-CuMTS3 ----------

Cultivate these samples in 3mL of LB medium with ChlPhe at 30°C overnight.

September 21, 2014

KOBAYASHI
Miniprep
Sample
Host Vector Insert Primer
DH5α(F) pSB1C3 muta-CuMTS3
DH5α(F) pSB1C3 muta-CuMTS3
DH5α(F) pSB1C3 muta-CuMTS3


Restriction Digest
Sample Enzyme Buffer
Marker Host Vector Insert Primer
1kb Ladder
pSB1C3 BBa_J04450 EcoR1 Buffer H
pSB1C3 BBa_J04450 Pst1 Buffer H
pSB1C3 BBa_J04450 EcoR1, Pst1 Buffer H
pSB1C3 BBa_J04450 Spe1 FD buffer
pSB1C3 BBa_J04450 Xba1 FD buffer
pSB1C3 BBa_J04450 Spe1, Xba1 FD buffer
pSB1C3 BBa_J04450
λDNA-HindⅢ
F pSB1C3 muta-CuMTS3 EcoR1 Buffer H
F pSB1C3 muta-CuMTS3 Pst1 Buffer H
F pSB1C3 muta-CuMTS3 EcoR1, Pst1 Buffer H
F pSB1C3 muta-CuMTS3 Spe1 FD buffer
F pSB1C3 muta-CuMTS3 Xba1 FD buffer
F pSB1C3 muta-CuMTS3 Spe1, Xba1 FD buffer
F pSB1C3 muta-CuMTS3

AGE
Lane Sample Enzyme
Marker Host Vector Insert Primer
1 1kb Ladder
2 pSB1C3 BBa_J04450 EcoR1
3 pSB1C3 BBa_J04450 Pst1
4 pSB1C3 BBa_J04450 EcoR1, Pst1
5 pSB1C3 BBa_J04450 Spe1
6 pSB1C3 BBa_J04450 Xba1
7 pSB1C3 BBa_J04450 Spe1, Xba1
8 pSB1C3 BBa_J04450
9 λDNA-HindⅢ
10 F pSB1C3 muta-CuMTS3 EcoR1
11 F pSB1C3 muta-CuMTS3 Pst1
12 F pSB1C3 muta-CuMTS3 EcoR1, Pst1
13 F pSB1C3 muta-CuMTS3 Spe1
14 F pSB1C3 muta-CuMTS3 Xba1
15 F pSB1C3 muta-CuMTS3 Spe1, Xba1
16 F pSB1C3 muta-CuMTS3


October

October 1, 2014

KOBAYASHI
Protein Extraction (S. cerevisiae)
Sample
Host Vector Insert Primer
BY4247 p427-TEF
BY4247 p427-TEF CuMTSE2 pGEX6P-2F1, pGEX6P-2R
BY4247 p427-TEF CuMTS3 pGEX6P-2F2, pGEX6P-2R
BY4247 p427-TEF CuMTS62 pGEX6P-2F2, pGEX6P-2R


SDS-PAGE
Lane Sample
Host Vector Insert Primer
1 BY4247 p427-TEF
2 BY4247 p427-TEF CuMTSE2 pGEX6P-2F1, pGEX6P-2R
3 BY4247 p427-TEF CuMTS3 pGEX6P-2F2, pGEX6P-2R
4 BY4247 p427-TEF CuMTS62 pGEX6P-2F2, pGEX6P-2R

October 2, 2014

KOBAYASHI
Preculture
Sample
Host Vector Insert Primer
BY4247 p427-TEF
BY4247 p427-TEF CuMTS3 pGEX6P-2F2, pGEX6P-2R

Cultivate these samples in 3mL of SD medium with G418 at 30°C overnight.

Western blot
Lane Sample
Host Vector Insert Primer
1 BY4247 p427-TEF
2 BY4247 p427-TEF CuMTSE2 pGEX6P-2F1, pGEX6P-2R
3 BY4247 p427-TEF CuMTS3 pGEX6P-2F2, pGEX6P-2R
4 BY4247 p427-TEF CuMTS62 pGEX6P-2F2, pGEX6P-2R

October 3, 2014

KOBAYASHI
Main culture
Cultivate following samples in Big Scale with G418 at 28°C overnight in shaken culture.
Sample
Host Vector Insert Primer
BY4247 p427-TEF
BY4247 p427-TEF CuMTS3 pGEX6P-2F2, pGEX6P-2R


Restriction Digest
Sample Enzyme Buffer
Vector Insert Primer
pSB1C3 EcoR1, Spe1 Buffer H
pSB1C3 Xba1, Pst1 Buffer H
Linearized pSB1A3 EcoR1, Pst1 Buffer H


AGE
Lane Marker Sample Enzyme
Vector Insert Primer
1 1kb Ladder
2 pSB1C3 EcoR1, Spe1
3 pSB1C3 Xba1, Pst1
4 Linearized pSB1A3 EcoR1, Pst1


DNA Refinement 1
Sample Enzyme
Vector Insert Primer
pSB1C3 EcoR1, Spe1
pSB1C3 Xba1, Pst1
pSB1C3 Xba1, Pst1
Linearized pSB1A3 EcoR1, Pst1, Dpn1


Ligation
Vector Sample Enzyme
Insert1 Insert2
pSB1A3 EcoR1, Xba1, Spe1, Pst1
pSB1A3 EcoR1, Xba1, Spe1, Pst1

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

Transformation
Host Sample
Vector Insert1 Insert2
DH5α pSB1A3
DH5α pSB1A3

October 4, 2014

KOBAYASHI
Colony Isolation
Isolate each colony from the plates which were inoculated overnight.
Host Sample Section
Vector Insert Primer
DH5α pSB1A3 1-16
DH5α pSB1A3 a-p


Monoterpene Extraction
Host Sample
Vector Insert Primer
BY4247 p427-TEF
BY4247 p427-TEF CuMTS3 pGEX6P-2F2, pGEX6P-2R

Samples were stocked in -4℃.

Preculture
Host Sample
Vector Insert Primer
BY4247 p427-TEF CuMTSE2 pGEX6P-2F2, pGEX6P-2R
BY4247 p427-TEF CuMTS62 pGEX6P-2F2, pGEX6P-2R

Cultivate samples in 3mL of SD medium with G418 at 30°C overnight.

October 5, 2014

KOBAYASHI
Miniprep
Host Sample
Vector Insert
DH5α pSB1A3
DH5α pSB1A3


Restriction Digest and AGE
Host Sample Enzyme
Vector Insert
DH5α pSB1C3 EcoR1, Pst1
DH5α pSB1C3 EcoR1, Pst1
pSB1C3 K1448000 EcoR1, Pst1

October 8, 2014

KOBAYASHI
GC-MS analysis
Host Sample
Vector Insert Primer
BY4247 p427-TEF
BY4247 p427-TEF CuMTS3 pGEX6P-2F2, pGEX6P-2R

Preculture
Host Sample
Vector Insert Primer
BY4247 p427-TEF
BY4247 p427-TEF CuMTS3 pGEX6P-2F2, pGEX6P-2R

October 9, 2014

KOBAYASHI
Main Culture
Host Sample
Vector Insert Primer
BY4247 p427-TEF
BY4247 p427-TEF CuMTS3 pGEX6P-2F2, pGEX6P-2R

October 10, 2014

KOBAYASHI
GC Analysis
Host Sample
Vector Insert Primer
BY4247 p427-TEF
BY4247 p427-TEF CuMTS3 pGEX6P-2F2, pGEX6P-2R

For More Information: BBa_K1448000

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