Team:KIT-Kyoto/Notebook/Labnote

From 2014.igem.org

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<h2>July</h2>
<h2>July</h2>
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<h3>7/25</h3>
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<!-- タイトル -->
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<h3>July 1, 2014</h3>
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<!-- タイトル終わり -->
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<!-- 本文開始 -->
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<p class="sentence">
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<!-- 実験者名 -->
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KOBAYAShI<br>
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<!-- 実験者名終わり -->
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<!--Protein Extraction (Saccharomyces cerevisiae)-->
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<strong>Protein Extraction (Saccharomyces cerevisiae)</strong>
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Divide each of 2 plates into 8 sections and inoculate following samples on them.
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<table class="materials">
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<tr>
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<th rowspan="2">Host</th>
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<th colspan="3">Sample</th>
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</tr>
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<tr>
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<th>Vector</th>
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<th>Insert</th>
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<th>Primer</th>
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</tr>
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<tr>
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<td>BY4247</td>
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<td>p427-TEF</td>
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<td>CuMTSE1</td>
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<td>P-F1,P-R</td>
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</tr>
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<tr>
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<td>BY4247</td>
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<td>p427-TEF</td>
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<td>CuMTSE1</td>
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<td>P-F2,P-R</td>
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</tr>
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</table>
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<!--Protein Extraction (Saccharomyces cerevisiae)-->
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<br>
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<!-- タイトル -->
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<h3>July 2, 2014</h3>
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<!-- タイトル終わり -->
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<!-- 本文開始 -->
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<p class="sentence">
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<!-- 実験者名 -->
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ONISHI<br>
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<!-- 実験者名終わり -->
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<!--Miniprep-->
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<strong>Miniprep</strong>
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<br>
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<table class="materials">
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<tr>
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<th rowspan="2">Host</th>
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<th colspan="3">Sample</th>
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</tr>
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<tr>
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<th>Vector</th>
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<th>Insert</th>
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<th>Primer</th>
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</tr>
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<tr>
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<td>BY4247</td>
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<td colspan="3">p426-TEF</td>
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</tr>
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<tr>
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<td>BY4247</td>
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<td>p427-TEF</td>
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<td>CuMTSE1</td>
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<td>P-F1,P-R</td>
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</tr>
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<tr>
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<td>BY4247</td>
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<td>p427-TEF</td>
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<td>CuMTSE1</td>
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<td>P-F2,P-R</td>
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</tr>
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</table>
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<!--Miniprep-->
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<h3>7/25</h3>
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<h2>August</h2>
<h2>August</h2>

Revision as of 18:53, 2 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)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
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 12th.

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 CuMTSE62 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 14th and June 11th.


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 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
Vector Insert
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
Vector Insert
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
Vector Insert
BL21(DE3)pLysS pGEX6P-2 CuMTSE11
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

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