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August
August 11th
Restriction Enzyme Digestion
sample | restriction enzymes |
His-RFP(with stop codon) | SpeI,EcoRI |
5'-intron M | SpeI,EcoRI |
Lac-3'intron-RBS | SpeI,EcoRI |
His-RFP(without stop codon) | XbaI,EcoRI |
RBS-Amp | SpeI,EcoRI |
Ligation
- EcoRI-His-RFP(with stop codon)-SpeI + XbaI-DT-Cm-EcoRI
- EcoRI-5'intron M-SpeI + XbaI-DT-Cm-EcoRI
- EcoRI-Lac-3'intron-RBS-SpeI + XbaI-His-RFP(without stop codon)-5'intron-DT-Cm-EcoRI
- EcoRI-His-RFP(without stop codon)-SpeI + SpeI-Amp-EcoRI
Transformation
- -His-RFP(with stop codon)-DT-Cm-
- -5'intron M-DT-Cm-
- -Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm-
- -His-RFP(without stop codon)-Amp-
Densitometry
sample | concentration(ng/µL) |
His-RFP(with stop codon) EcoRI-SpeI | 162.6 |
5'intron M EcoRI-SpeI | 45.4 |
Lac-3'intron-RBS EcoRI-SpeI | 120.9 |
His-RFP(without stop codon) EcoRI-SpeI | 160.0 |
His-RFP(without stop codon)-5'intron-DT-Cm XbaI-EcoRI |
174.0 |
Amp EcoRI-SpeI | 101.8 |
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August 12th
Colony PCR
- His-RFP(with stop codon)-DT-Cm
- 5'intron M-DT-Cm
- Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm:[1]turning red
- Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm:[2]turning pink
- Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm:[3]turning white
- His-RFP(without stop codon)-Amp
Electrophoresis
- His-RFP(with stop codon)-DT
- His-RFP
- 5'intron M-DT
- 5'intron M
- Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT:[1]
- Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT:[2]
- Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT:[3]
- His-RFP(without stop codon)-5'intron-DT
- His-RFP(without stop codon)
Liquid Culture
- His-RFP(with stop codon)-DT
- 5'intron M-DT
- Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT:[1]
- Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT:[3]
- His-RFP(without stop codon)
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August 13th
Miniprep
PCR
By use of KAPA HiFi DNA Polymerase
- His-RFP(with stop codon)-DT-Cm
- 5'intron M-DT-Cm
By use of Taq DNA Polymerase
- Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm:[1]turning red
- Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm:[3]turning white
Electrophoresis
- His-RFP(with stop codon)-DT-Cm
- 5'intron M-DT-Cm
- DNA Ladder Markers DNA Ladder One(Broad Range)
- Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm:[1]turning red
- Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm:[3]turning white
Restriction Enzyme Digestion
sample | restriction enzymes |
His-RFP(with stop codon)-DT-Cm | EcoRI,XbaI |
5'intron M-DT-Cm | EcoRI,XbaI |
Densitometry
sample | concentration(ng/µL) |
EcoRI-Lac-RBS-SpeI | 65.0 |
XbaI-His-RFP(with stop codon)-DT-Cm-EcoRI | 188.8 |
EcoRI-His-RFP(with stop codon)-SpeI | 160.0 |
EcoRI-His-RFP(without stop codon)-SpeI | 160.0 |
XbaI-5'intron M-DT-Cm-EcoRI | 167.8 |
Ligation
- EcoRI-Lac-RBS-SpeI + XbaI-His-RFP(with stop codon)-DT-Cm-EcoRI
- EcoRI-His-RFP(with stop codon)-SpeI + XbaI-5'intron M-DT-Cm-EcoRI
- EcoRI-His-RFP(without stop codon)-SpeI + XbaI-5'intron M-DT-Cm-EcoRI
Transformation
- -Lac-RBS-His-RFP(with stop codon)-DT-Cm-
- -His-RFP(with stop codon)-5'intron M-DT-Cm-
- -His-RFP(without stop codon)-5'intron M-DT-Cm-
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August 16th
Liquid Culture
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August 18th
Plasmid Extraction
- -Lac-RBS-His-RFP(with stop codon)-DT-Cm-
- -His-RFP(with stop codon)-5'intron M-DT-Cm-
- -His-RFP(without stop codon)-5'intron M-DT-Cm-
Plasmid Purification
- -5'intron M-His-RFP(with stop codon)-
- -5'intron M-His-RFP(without stop codon)-
Miniprep
Colony PCR
Restriction Enzyme Digestion
sample | restriction enzymes |
-His-RFP(with stop codon)-5'intron M-DT-Cm- | EcoRI,XbaI |
-His-RFP(without stop codon)-5'intron M-DT-Cm- | EcoRI,XbaI |
Densitometry
sample | concentration(ng/µL) |
EcoRI-Lac-3'intron-RBS-SpeI | 120.0 |
XbaI-His-RFP(with stop codon)-5'intron M-DT-Cm-EcoRI | 187.8 |
XbaI-His-RFP(without stop codon)-5'intron M-DT-Cm-EcoRI | 158.9 |
Ligation&Transformation
- EcoRI-Lac-3'intron-RBS-SpeI + XbaI-His-RFP(with stop codon)-5'intron M-DT-Cm-EcoRI
- EcoRI-Lac-3'intron-RBS-SpeI + XbaI-His-RFP(without stop codon)-5'intron M-DT-Cm-EcoRI
Polyethylene glycol Precipitation
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August 19th
Colony PCR
- -Lac-3'intron-RBS-His-RFP(with stop codon)-5'intron M-DT-Cm-
- -Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron M-DT-Cm-
Electrophoresis
- -Lac-3'intron-RBS-His-RFP(with stop codon)-5'intron M-DT-Cm-
- -Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron M-DT-Cm-
- marker
- -His-RFP(with stop codon)-5'intron M-DT-Cm-
- -His-RFP(without stop codon)-5'intron M-DT-Cm-
Shaking Culture
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August 20th
Colony PCR
- -Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron M-DT-Cm-
by use of another colony differs from 8/19
Electrophoresis
- -Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron M-DT-Cm-
- marker
- -His-RFP(without stop codon)-5'intron M-DT-Cm-
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August 21th
Reagent Preparation
Protein Extraction
SDS-PAGE
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August 22th
Prorein Purification by use of Ni-NTA
SDS-PAGE
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August 23th
Prorein Purification by use of Ni-NTA
SDS-PAGE
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August 24th
Extraction of plasmid device
- -Lac-RBS-His-RFP(with stop codon)-DT-Cm-
- -Lac-RBS-3'intron-His-RFP(without stop codon)-5'intron-DT-Cm-
- -Lac-RBS-3'intron-His-RFP(with stop codon)-5'intron M-DT-Cm-
- -Lac-RBS-3'intron-His-RFP(without stop codon)-5'intron M-DT-Cm-
PCR&Electrophoresis
- EcoRI-His-RFP(with stop codon)-PstI
- EcoRI-His-RFP(without stop codon)-PstI
- EcoRI-5'intron M-PstI
- EcoRI-Lac-3'intron-RBS-PstI
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August 25th
Extraction of plasmid device
SDS-PAGE
- Lac-3'intron-RBS-His-RFP-5'intron M-DT-Cm
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August 26th
PCR
Restriction Enzyme Digestion
sample | restriction enzymes |
His-RFP(with stop codon) | EcoRI,PstI |
His-RFP(without stop codon) | EcoRI,PstI |
5'intron M | EcoRI,PstI |
Lac-3'intron-RBS | EcoRI,PstI |
TTHA0175 | EcoRI,SpeI |
Densitometry
sample | concentration(ng/µL) |
His-RFP(with stop codon) | 180.7 |
His-RFP(without stop codon) | 164.6 |
5'intron M | 37.7 |
Lac-3'intron-RBS | 107.5 |
TTHA0175 | 112.7 |
Ligation&Transformation
- EcoRI-His-RFP(with stop codon)-PstI + EcoRI-Cm-PstI
- EcoRI-His-RFP(without stop codon)-PstI + EcoRI-Cm-PstI
- EcoRI-5'intron M-PstI + EcoRI-Cm-PstI
- EcoRI-Lac-3'intron-RBS-PstI + EcoRI-Cm-PstI
- EcoRI-TTHA0175-SpeI + EcoRI-5'intron M-DT-Cm-XbaI
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August 27th
Colony PCR
- -His-RFP(with stop codon)-Cm-
- -TTHA0175-Amp-
- -TTHA0175-5'intron M-DT-Cm-
Shaking Culture
- -His-RFP(with stop codon)-Cm-
- -TTHA0175-Amp-
- -TTHA0175-5'intron M-DT-Cm-
Enrichment Culture
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August 28th
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