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 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


July

7/25



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August




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October




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