Material&Methods

• 1) Parts
• 2) Primer list
• 3) Materials
• 3-1 Kit
• 3-2 Restriction Enzyme
• 3-3 Polymerase
• 3-4 DNA ligase
• 3-5 Marker
• 3-6 Organism
• 3-7 Antibiotics
• 3-8 Equipment
• 3-9 Backbone
• 3-10 Buffer
• 3-11 SDS-PAGE&Westernblotting
• 4) Methods
• 4-1 Miniprep
• 4-2 Gel Extraction and PCR purification
• 4-3 Restriction Enzyme Digestion
• 4-4 Ligation
• 4-5 Transformation
• 4-6 PCR
• 4-7 Sequencing
• 4-8 Western blotting
• 4-9 Overnight-Culture
• 4-10 Plates

1) Parts

Basic Parts

Composite Parts

2) Primer List

3) Materials

3-1 Kit

Name	Supplier
FastGene™Plasmid Mini Kit	NIPPON Genetics Co., Ltd
FastGene™Gel/PCR Extraction Kit	NIPPON Genetics Co.,Ltd

3-2 Restriction Enzyme

Name	Supplier
EcoRI	TaKaRa, Promega
PstI	TaKaRa, Promega
SpeI	TaKaRa, Promega

3-3 Polymerase

Name	Supplier
Q5 High-Fidelity 2x master mix	New England Biolabs Japan Inc.

3-4 DNA ligase

Name	Supplier
T4 DNA ligase	TaKaRa

3-5 Marker

Name	Supplier
1kb DNA Ladder	NIPPON Genetics Co., Ltd

3-6 Organism

Name	Supplier
E.coli DH5α Genotype	TaKaRa

3-7 Antibiotics

Name	Supplier
Chloramphenicol	Wako
Ampicillin	Wako

3-8 Equipment

Name	Supplier
BioPhotometer	eppendorf
LABO SHAKER	BIO CRAFT
CO2 INCUBATOR	SANYO
Pipette Controller	Biohit Midi Plus
MiniCentrifuge Model GMC-060	LMS CO.,LTD.
HIGH-SPEED REFRIGERATED CENTRIFUGE	TOMY
HIGH-PRESSURE STEAM STERILIZER	TOMY
VOTEX-GENE2	Scientific Industryes
Scanning Electron Miniscope TM1000s	HITACHI
Fluorescence Microscope BX61N-34-FL-1-D	OLYMPUS
SCIECE IMAGING SYSTEM LAS-3000	Fuji film

3-9 Backbone

Name	Supplier
pRS421	National BioResource Project http://yeast.nig.ac.jp/yeast/
pRS423	National BioResource Project
pRS424	National BioResource Project
pRS425	National BioResource Project
pRS426	National BioResource Project
pGK421	National BioResource Project
pRS316	National BioResource Project

3-10 Buffer

Name	Supplier
Sodium Carbonate	Wako
Sodium Bicarbonate	Wako
Methanol(99.5%)	Wako
Sodium Chloride	Wako
SDS	nacalai tesque
Trisaminomethane	nacalai tesque
Potassium dihydrogenphosphste	SIGAMA-ALDRICH
Potassium Chloride	Wako
Glycine	Wako
Agar, Powder	Wako
Agarose XP	Wako
10% Hydrochloric Acid	Wako

3-11 SDS-PAGE&Westernblotting

Name	Supplier
REAL GEL PLATE (10%)	BIO CRAFT
BE-210(SDS-PAGE)	BIO CRAFT
BE-330	BIO CRAFT
Amersham ECL Anti-Mouse IgG	GE healthcare
Amersham ECL Anti-rabbit IgG	GE healthcare
Amersham ECL Prime Blocking Reagent	GE healthcare
Amersham ECL Prime WB Detection Reagent	GE healthcare

4) Methods

4-1 Miniprep

Minipreps were performed using FastGene™Plasmid Mini Kit according to the manufacturer's protocols.

4-2 Gel Extraction and PCR purification

Gel Extraction was performed using FastGene™Gel/PCR Extraction Kit according to the manufacturer's protocols.

4-3 Restriction Enzyme Digestion

Restriction enzyme treatment was performed using Q5 High-Fidelity 2x master mix according to the respective manufacturer's protocols.

4-4 Ligation

Ligation was performed using Takara Bio's Ligation High Ver.2 according to the manufacturer's.

4-5 Transformation

1.Thaw competent cells on ice
2.Add DNA sample (2.5μl) to competent cells. leave them on ice for 15 minutes
3.Keep them in heating block for 45 seconds, then cool on ice for 2 minutes
4.Add 90μl SOC liquid culture medium, then incubate for 45 minutes at 37℃
5.Seed cells onto LB+antibiotics agar plate. Incubate for 24 hours at 37℃

4-6 PCR

PCR was performed using Q5 High-Fidelity 2x master mix according to the manufacturer's protocols.

4-7 Sequencing

We outsourced the sequencing to Macrogen

http://www.macrogen-japan.co.jp/cap_seq_0203.php

4-8 Western blotting

1.Apply sample to SDS-PAGE minigel (BIOCRAFT 15%).
2.Soak the gel in transfer buffer.
3.Soak PVDF membrane in 100% methanol for 30 sec.
4.Soak PVDF membrane and filter paper in transfer buffer, leave for 5 min.
5.Set filter paper, membrane, gels, filter paper in this order to semidry transfer from anode to cathode.
6.Transfer the proteins from the gel to the membrane with 100 mA for 1 h.
7.Soak membrane in blocking buffer (dilution of 5 g ECL Blocking reagent by 100ml of TBST) for 1 h.
8.Wash for 5 min 3 times with TBST.
9.Apply 1' antibody (dilution of 10 μl of 1’antibody by 10ml of Blocking buffer) and shake for 1 h.
10.Wash for 5 min 3 times with TBST.
11.Apply 2' antibody (dilution of 4μl of 2’antibody by 10ml of Blocking buffer) and shake for 1 h.
12.Wash for 5 min 3 times with TBST.
13.Drain excess wash buffer from the washed membrane and place on flat surface, protein side up.
14.Add detection reagent onto the membrane, covering all of the membrane.
15.Incubate for 5 minutes at room temperature.
16.Drain off excess detection reagent by dabbing with Kimwipe.
17.Place the sample in the CCD camera compartment and record the images.

4-9 Overnight-Culture

1.Prepare LB media by resolving 5g Bacto Tryptone, 1.25g Bacto-yeast extract and 1.25g NaCl in 500ml of milliQ and autoclave it.
2.Transfer 7ml of LB into a culture tube and add the respective antibiotic stock solutions.
3.Inoculate the culture by picking a single colony with a pipet tip and tipping it into the medium.
4.Shake the culture at 37℃ at 180 rpm for 14-16h.
5.Isolate plasmids or use the culture for further experiments.

Concentrations of antibiotics:

Antibiotics	Stock concentration	Final concentration
Ampicillin	100mg/ml	100μg/ml
Chloramphenicol	10mg/l	30μg/ml

4-10 Plates

1.Rinse the graduated cylinder with distilled water and add 500 ml of milliQ.
2. Transfer 1. to the flask.
3. Place the following in the flask of 2.
・Bacto Tryptone 5 g
・Bacto yeast extract 2.5 g
・NaCl 2.5g
・Agar 7.5 g
4. After mixing thoroughly, autoclave it after covering with aluminum foil.
5. Add the respective antibiotics.
6. Cool until it gets moderately warm to the touch.
7. Put in a container and fix the lid with the tape after the medium solidifies.

Concentrations of antibiotics:

Antibiotics	Stock concentration	Final concentration
Ampicillin	100mg/ml	100μg/ml
Chloramphenicol	10mg/ml	30μg/ml