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Protocols Calender

Calender

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August
Mon	Tue	Wed	Thr	Fri	Sat	Sun
				1	2	3
4	5	6	7	8	9	10
11	12	13	14	15	16	17
18	19	20	21	22	23	24
25	26	27	28	29	30	31

September
Mon	Tue	Wed	Thr	Fri	Sat	Sun
1	2	3	4	5	6	7
8	9	10	11	12	13	14
15	16	17	18	19	20	21
22	23	24	25	26	27	28
29	30

October
Mon	Tue	Wed	Thr	Fri	Sat	Sun
		1	2	3	4	5
6	7	8	9	10	11	12
13	14	15	16	17	18	19
20	21	22	23	24	25	26
27	28	29	30	31

Glossary

His

alphabet

GFP

green fluorescence protein

RFP

red fluorescent protein

5'-intron

5'-intron M

3'-intron

Lac

Amp

ampicillin resistance gene

Cm

chloramphenicol resistance gene

RBS

DT

EcoRI

restriction site that is cut by EcoRI or restriction enzyme

XbaI

restriction site that is cut by XbaI or restriction enzyme

SpeI

restriction site that is cut by SpeI or restriction enzyme

PstI

restriction site that is cut by PstI or restriction enzyme

TTHA0715

cold shock protein with amino acid sequence cut by thrombin

TTHA0715 guanidine deposition

SmtA

circular RNA

Prologue -before August-

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

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

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

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

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

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

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

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

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

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

sample	concentration(ng/µL)
EcoRI-TTHA0175-5'intron M-DT-Cm-XbaI	118.8

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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September 3rd

Culture

1. -Lac-3'intron-RBS-RFP(without stop codon)-5'intron M-DT-Cm- 20 mL
2. -Lac-3'intron-RBS-RFP(with stop codon)-5'intron M-DT-Cm- 5 mL

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September 4th

Make circular RNA

1. TTHA0715
2. RFP(without stop codon)
3. RFP(with stop codon)

Cell body Crush

1. TTHA0715
2. RFP(without stop codon)
3. RFP(with stop codon)

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September 5th

SDS-PAGE

1. GFP
2. TTHA0715 supernatant1(no wash)
3. TTHA0715 supernatant2(first wash)
4. TTHA0715 supernatant3(second wash)
5. TTHA0715 deposition
6. TTHA0715 deposition two fold dilution
7. marker
8. RFP(without stop codon) supernatant1
9. RFP(without stop codon) deposition
10. RFP(with stop codon) supernatant1
11. RFP(with stop codon) deposition
12. RFP(without stop codon) supernatant2
13. RFP(without stop codon) supernatant3
14. RFP(with stop codon) supernatant2
15. RFP(with stop codon) supernatant3

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September 8th

SDS-PAGE

1. marker
2. supernatant
3. refined RFP

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September 9th

HisTag refining

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September 10th

SDS-PAGE with 10% gel

1. TTHA0715 deposition
2. TTHA0715 supernatant
3. marker
4. RFP and fungus bodies
5. dialyzed RFP
6. guanidine deposition

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September 11th

SDS-PAGE

1. culture supernatant on September 1st
2. TTHA0715 supernatant on September 1st
3. TTHA0715 deposition on September 1st
4. marker
5. TTHA0715 supernatant on September 4th
6. TTHA0715 deposition on September 4th
7. TTHA0715 guanidine deposition

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September 12th

Ligation

1. promoter + RBS-GFP-DT-Cm
2. TTHA0715 + 5'intron-DT-Cm
3. Lac + RBS-GFP-DT-Cm

Transformation

1. -promoter-RBS-GFP-DT-Cm-
2. -Lac-3'intron-RBS-TTHA0715-5'intron-DT-Cm-
3. -BBa_K519010-pSB1C3-
4. -Lac-RBS-GFP-DT-Cm-

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September 13th

RNA Extraction

1. circular RNA (RFP without stop codon)

Densitometry

1. circular RNA (RFP without stop codon)

DNase treatment

RNase treatment

reverse transcription PCR

cDNA synthesis

PCR

koujityu

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