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Team:KIT-Kyoto/Notebook/Labnote
From 2014.igem.org
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 | 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 | CuMTS3 | Sal1, Not1 | |
| 11 | pGEX6P-2 | CuMTS62 | N.D | |
| 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 | Enzyme | |
|---|---|---|---|---|
| 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 | |
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 | 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
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 | 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 Refinement2
| 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 |
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 | N.D | |||
| 6 | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | |||
| 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 | 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 |
July
July 1, 2014
KOBAYASHI
Protein Extraction (Saccharomyces 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 | N.D | |||
| 2 | p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | N.D | |
| 3 | p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | N.D | |
| 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 (Saccharomyces 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 | CuMTSE2 | pGEX6P-2F2, pGEX6P-2R |
July 6, 2014
SOGA
DNA Refinement1
| 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 Refinement2
| 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 | N.D |
| 2 | p427-TEF(ア) | CuMTS3 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 |
| 3 | p427-TEF(イ) | CuMTS3 | pGEX6P-2F1, pGEX6P-2R | N.D |
| 4 | p427-TEF(イ) | CuMTS3 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 |
| 5 | p427-TEF(エ) | CuMTS3 | pGEX6P-2F1, pGEX6P-2R | N.D |
| 6 | p427-TEF(エ) | CuMTS3 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 |
| 7 | p427-TEF(d) | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | N.D |
| 8 | p427-TEF(d) | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 |
| 9 | p427-TEF(h) | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | N.D |
| 10 | p427-TEF(h) | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 |
| 11 | p427-TEF(l) | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | N.D |
| 12 | p427-TEF(l) | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 |
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