Team:Gifu/Notebook

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AugustSeptember
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Glossary

AAA
alphabet

August

August 11th

Restriction Enzyme Digestion

samplerestriction enzymes
His-RFP(with stop codon)SpeI,EcoRI
5'-intron MSpeI,EcoRI
Lac-3'intron-RBSSpeI,EcoRI
His-RFP(without stop codon)XbaI,EcoRI
RBS-AmpSpeI,EcoRI

Ligation

  1. EcoRI-His-RFP(with stop codon)-SpeI + XbaI-DT-Cm-EcoRI
  2. EcoRI-5'intron M-SpeI + XbaI-DT-Cm-EcoRI
  3. EcoRI-Lac-3'intron-RBS-SpeI + XbaI-His-RFP(without stop codon)-5'intron-DT-Cm-EcoRI
  4. EcoRI-His-RFP(without stop codon)-SpeI + SpeI-Amp-EcoRI

Transformation

  1. -His-RFP(with stop codon)-DT-Cm-
  2. -5'intron M-DT-Cm-
  3. -Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm-
  4. -His-RFP(without stop codon)-Amp-

Densitometry

sampleconcentration(ng/µL)
His-RFP(with stop codon) EcoRI-SpeI162.6
5'intron M EcoRI-SpeI45.4
Lac-3'intron-RBS EcoRI-SpeI120.9
His-RFP(without stop codon) EcoRI-SpeI160.0
His-RFP(without stop codon)-5'intron-DT-Cm XbaI-EcoRI 174.0
Amp EcoRI-SpeI101.8

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August 12th

Colony PCR

  1. His-RFP(with stop codon)-DT-Cm
  2. 5'intron M-DT-Cm
  3. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm:[1]turning red
  4. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm:[2]turning pink
  5. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm:[3]turning white
  6. His-RFP(without stop codon)-Amp

Electrophoresis

  1. His-RFP(with stop codon)-DT
  2. His-RFP
  3. 5'intron M-DT
  4. 5'intron M
  5. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT:[1]
  6. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT:[2]
  7. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT:[3]
  8. His-RFP(without stop codon)-5'intron-DT
  9. His-RFP(without stop codon)

Liquid Culture

  1. His-RFP(with stop codon)-DT
  2. 5'intron M-DT
  3. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT:[1]
  4. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT:[3]
  5. His-RFP(without stop codon)

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August 13th

Miniprep

PCR

By use of KAPA HiFi DNA Polymerase

  1. His-RFP(with stop codon)-DT-Cm
  2. 5'intron M-DT-Cm

By use of Taq DNA Polymerase

  1. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm:[1]turning red
  2. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm:[3]turning white

Electrophoresis

  1. His-RFP(with stop codon)-DT-Cm
  2. 5'intron M-DT-Cm
  3. DNA Ladder Markers DNA Ladder One(Broad Range)
  4. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm:[1]turning red
  5. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm:[3]turning white

Restriction Enzyme Digestion

samplerestriction enzymes
His-RFP(with stop codon)-DT-CmEcoRI,XbaI
5'intron M-DT-CmEcoRI,XbaI

Densitometry

sampleconcentration(ng/µL)
EcoRI-Lac-RBS-SpeI65.0
XbaI-His-RFP(with stop codon)-DT-Cm-EcoRI188.8
EcoRI-His-RFP(with stop codon)-SpeI160.0
EcoRI-His-RFP(without stop codon)-SpeI160.0
XbaI-5'intron M-DT-Cm-EcoRI167.8

Ligation

  1. EcoRI-Lac-RBS-SpeI + XbaI-His-RFP(with stop codon)-DT-Cm-EcoRI
  2. EcoRI-His-RFP(with stop codon)-SpeI + XbaI-5'intron M-DT-Cm-EcoRI
  3. EcoRI-His-RFP(without stop codon)-SpeI + XbaI-5'intron M-DT-Cm-EcoRI

Transformation

  1. -Lac-RBS-His-RFP(with stop codon)-DT-Cm-
  2. -His-RFP(with stop codon)-5'intron M-DT-Cm-
  3. -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

  1. -Lac-RBS-His-RFP(with stop codon)-DT-Cm-
  2. -His-RFP(with stop codon)-5'intron M-DT-Cm-
  3. -His-RFP(without stop codon)-5'intron M-DT-Cm-

Plasmid Purification

  1. -5'intron M-His-RFP(with stop codon)-
  2. -5'intron M-His-RFP(without stop codon)-

Miniprep

Colony PCR

Restriction Enzyme Digestion

samplerestriction 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

sampleconcentration(ng/µL)
EcoRI-Lac-3'intron-RBS-SpeI120.0
XbaI-His-RFP(with stop codon)-5'intron M-DT-Cm-EcoRI187.8
XbaI-His-RFP(without stop codon)-5'intron M-DT-Cm-EcoRI158.9

Ligation&Transformation

  1. EcoRI-Lac-3'intron-RBS-SpeI + XbaI-His-RFP(with stop codon)-5'intron M-DT-Cm-EcoRI
  2. 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

  1. -Lac-3'intron-RBS-His-RFP(with stop codon)-5'intron M-DT-Cm-
  2. -Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron M-DT-Cm-

Electrophoresis

  1. -Lac-3'intron-RBS-His-RFP(with stop codon)-5'intron M-DT-Cm-
  2. -Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron M-DT-Cm-
  3. marker
  4. -His-RFP(with stop codon)-5'intron M-DT-Cm-
  5. -His-RFP(without stop codon)-5'intron M-DT-Cm-

Shaking Culture

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August 20th

Colony PCR

  1. -Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron M-DT-Cm-

by use of another colony differs from 8/19

Electrophoresis

  1. -Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron M-DT-Cm-
  2. marker
  3. -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

  1. -Lac-RBS-His-RFP(with stop codon)-DT-Cm-
  2. -Lac-RBS-3'intron-His-RFP(without stop codon)-5'intron-DT-Cm-
  3. -Lac-RBS-3'intron-His-RFP(with stop codon)-5'intron M-DT-Cm-
  4. -Lac-RBS-3'intron-His-RFP(without stop codon)-5'intron M-DT-Cm-

PCR&Electrophoresis

  1. EcoRI-His-RFP(with stop codon)-PstI
  2. EcoRI-His-RFP(without stop codon)-PstI
  3. EcoRI-5'intron M-PstI
  4. EcoRI-Lac-3'intron-RBS-PstI

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August 25th

Extraction of plasmid device

SDS-PAGE

  1. Lac-3'intron-RBS-His-RFP-5'intron M-DT-Cm

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August 26th

PCR

Restriction Enzyme Digestion

samplerestriction enzymes
His-RFP(with stop codon)EcoRI,PstI
His-RFP(without stop codon)EcoRI,PstI
5'intron MEcoRI,PstI
Lac-3'intron-RBSEcoRI,PstI
TTHA0175EcoRI,SpeI

Densitometry

sampleconcentration(ng/µL)
His-RFP(with stop codon)180.7
His-RFP(without stop codon)164.6
5'intron M37.7
Lac-3'intron-RBS107.5
TTHA0175112.7

Ligation&Transformation

  1. EcoRI-His-RFP(with stop codon)-PstI + EcoRI-Cm-PstI
  2. EcoRI-His-RFP(without stop codon)-PstI + EcoRI-Cm-PstI
  3. EcoRI-5'intron M-PstI + EcoRI-Cm-PstI
  4. EcoRI-Lac-3'intron-RBS-PstI + EcoRI-Cm-PstI
  5. EcoRI-TTHA0175-SpeI + EcoRI-5'intron M-DT-Cm-XbaI

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August 27th

Colony PCR

  1. -His-RFP(with stop codon)-Cm-
  2. -TTHA0175-Amp-
  3. -TTHA0175-5'intron M-DT-Cm-

Shaking Culture

  1. -His-RFP(with stop codon)-Cm-
  2. -TTHA0175-Amp-
  3. -TTHA0175-5'intron M-DT-Cm-

Enrichment Culture

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August 28th

Plasmid Purification

  1. -His-RFP(with stop codon)-5'intron M-Cm-
  2. -TTHA0175-5'intron M-DT-Cm-

PCR

  1. -His-RFP(with stop codon)-Cm-
  2. -Lac-3'intron-RBS-Cm-
  3. -His-RFP(without stop codon)-Cm-
  4. -5'intron M-Cm-

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August 29th

Densitometry

sampleconcentration(ng/µL)
EcoRI-TTHA0175-5'intron M-DT-Cm-XbaI118.8

Ligation

  1. EcoRI-TTHA0175-5'intron M-DT-Cm-XbaI + EcoRI-Lac-3'intron-RBS-SpeI

SDS-PAGE

Electrophoresis

  1. EcoRI-TTHA0175-5'intron M-DT-Cm-XbaI

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August 30th

Colony PCR

  1. -Lac-3'intron-RBS-TTHA0715-5'intron M-DT-Cm-
  2. Plate4 3G

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August 31th

Colony PCR

Because result of Colony PCR on 8/30 is not good, We chose 5 colonies and put them on PCR.

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September 1st

Shaking Culture

  1. -Lac-3'intron-RBS-TTHA0715-5'intron M-DT-Cm-

Cell body Crush&SDS-PAGE(gel 10%)

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