Difference between revisions of "Team:Kyoto/Experiments"

Materials

Kit	Supplier
Wizard® SV Gel and PCR	Promega
Ligation high Ver.2	TOYOBO
FastGene™Plasmid Mini Kit	NIPPON Genetics Co.,Ltd

Enzyme	Supplier
EcoRI	TaKaRa, Promega
PstI	TaKaRa, Promega
SpeI	TaKaRa, Promega
XbaI	TaKaRa, Promega
DraII	TaKaRa

Marker	Supplier
1kb DNA Ladder	TaKaRa

Organism	Supplier
E.coli DH5α Genotype	TaKaRa
E.coli JM109 Competent Cells	TaKaRa

Antibiotics	Supplier
Chloramphericol	nacalai tesque
Ampicillin Sodium Salt	nacalai tesque

Equipment Supplier

BEMLISE SR601	AsahiKASEI
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

Backbones	Supplier
pSB1C3	iGEM registry
pSB1A2	iGEM registry
pSB3C5	iGEM registry
BBa_ J61002	iGEM registry

Buffer	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
Tween-20	SANTA CRUZ

Other Enzyme	Supplier
KAPA™HiFi HotStart ReadyMix (2x)	KAPABIOSYSTEMS
KAPA2G™ Fast HotStart ReadyMix with dye (2x)	KAPABIOSYSTEMS
KAPATaq™EXtra HotStart ReadyMix with dye	KAPABIOSYSTEMS
Quick Taq® HS DyeMix	TOYOBO

SDSPAGE/WB	Supplier
IMMOBILON - P Blotting Sandwiches	Immobilon
REAL GEL PLATE (10%)	BIO CRAFT
BE-210(SDSPAGE)	BIO CRAFT
BE-330	BIO CRAFT
Amersham ECL Anti-Mouse IgG	GE healthcare
Amersham ECL Anti-rabbit IgG	GE healthcare
G2-4 rabbit anti-serum	Sano
Amersham ECL Prime Blocking Reagent	GE healthcare
Amersham ECL Prime WB Detection Reagent	GE healthcare

Methods

Westernblot (Against His tag)

1. Apply sample to SDS-PAGE minigel (BIOCLAFT 10%).
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 to semidry transferor in this order from anode to cathode
6. Transfer the proteins from the gels to the membrane with 100mA for 1 h
7. Soak membrane in blocking buffer (ECL Blocking reagent 5 g TBST 100ml) for 1 h
8. Wash for 5 min 3 times with TBST
9. Apply 1' antibody (anti-His tag or anti-NoVLP 10μl TBST 10 ml) and shake for 1 h
10. Wash for 5 min 3 times with TBST
11. Apply 2' antibody (anti-rabbit HRP 4μl:TBST 10ml) and shake for 1 h
12. Wash for 5 min 3 times
13. Drain excess wash buffer from the washed membrane and place it protein side up on flat surface
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

Western Blot (against NoVLP)

*Sample Preparation*

1. Mix milliQ 12.5ul, SDSlysisbuffer 4.5ul, and VLP.
2. Mix MilliQ 2.5ul, SDSlysisbuffer 2.5ul, and GE dual protein marker 5ul.
3. Vortex 1. and 2., then centrifuge them with a minicentrifuge.
4. Heat the solutions at 95 °C for 5 min.
5. Vortex the solutions, and centrifuge them again.

Cellulose Affinity Assay

1. Prepare LB +CP medium that contains objective E.coli, incubate overnight at 37°C
2. Centrifuge this solution at 200g for 10 min.
3. Collect the pellet, and add Carbonate bicarbonate buffe so that OD600 values come to about 0.2
4. Take 1ml out of the cell suspension, and measure OD600 values. This OD600 values are <before>.
5. Incubate the cell suspension with 3cm by 2.5 cm cellulose sheet (BEMLIESE #606) for 1 h
6. Measure OD600 values of the cell suspension. This OD600 values are <after>.
7. Transfer the cellulose sheet to a new buffer of the same volume, incubate for 1 hour
8. Measure OD600 values of the buffer This OD600 values are <wash>.

Growth Curve Drawing

1. Measure OD600 of LB liquid medium (10μg/ml CP) and set blank.
2. Prepare 10ml LB liquid medium (10μg/ml CP) with E.coli sample whose OD600 values are adjusted to 1.0, Cover the tube with a plastic sheet with airholes
3. Start incubating at 160rpm, 37°C. Measure the absorbance promptly and set it 0 minute.
4. Measure OD600 every 30 minutes (before 2hours)/every an hour (after 2hours).

RT-PCR

RT-PCR was performed using QuantiTect® Reverse Transcription Kit according to the manufacturer's protocol.

Scanning Electron Microscopy (2015 Summer Experiment)

Samples	Centrifugal Force
INPNC-His-scFv(pSB3C5) 25ul+VLP2.5ul	200g
BclA-His-scFv(pSB3C5)25ul+VLP2.5ul	200g

*Sample Preparation (E.coli+VLP)*

1. Prepare 45ml of sample E.coli liquid culture whose OD600 values are around 1.0
2. Take 25ul, and pour into sample tubes.
3. Centrifuge VLP with MiniCentrifuge. After tapping it, pour 2.5ul into tubes.
4. Incubate for 24 hours.
5. Centrifuge at 200g for 10 minutes.
6. Suspend the pellets in 1ml PBS.
7. Centrifuge at 200g for 10 minutes.
8. Remove half of the supernatant, then suspend it again.

Scanning Electron Microscopy (2016 Summer Experiment)

Samples	Centrifugal Force
INPNC-His-scFv(pSB3C5) 25ul+VLP2.5ul	3000g
BclA-His-scFv(pSB3C5)25ul+VLP2.5ul	3000g
INPNC-His-ctl 25ul+VLP2.5ul	3000g
BclA-His-ctl 25ul+VLP2.5ul	3000g
INPNC-His-scFv(pSB3C5) 25ul+VLP2.5ul	200g
BclA-His-scFv(pSB3C5)25ul+VLP2.5ul	200g
INPNC-His-ctl 25ul+VLP2.5ul	200g
BclA-His-ctl 25ul+VLP2.5ul	200g

*Sample Preparation (E.coli+VLP)*

1. Prepare 45ml of sample E.coli liquid culture whose OD600 values are adjusted to 1.0
2. Take 25ul, and pour into sample tubes.
3. Centrifuge VLP with MiniCentrifuge. After tapping it, pour 2.5ul into tubes.
4. Incubate for 24 hours.
5. Centrifuge at 200g/3000g for 10 minutes.
6. Suspend the pellets in 1ml PBS.
7. Centrifuge at 200g/3000g for 10 minutes.
8. Remove half of the supernatant, then suspend it again.

*Sample Preparation (PBS+VLP)*

1. After tapping VLP, pour 2ul into 200ml PBS.
2. Mark where pellets should have aggregated if E. coli were in the tube.
3. Centrifuge at 200g for 10 minutes.
4. Take 100ul of the supernatant, then pour into 1. tube.

Miniprep

Miniprep was performed using FastGene™Plasmid Mini Kit according to the manufacturer's protocols.

Gel Extraction

Gel Extraction was performed using FastGene™Plasmid Mini Kit(GP3→MilliQ), and　Wizard® SV Gel and PCR according to the manufacturer's protocols.

Restriction Enzyme Digestion

Restriction enzyme treatment was performed using Takara Bio's or Promega's restriction enzymes according to the respective manufacturer's protocols.

Ligation

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

Transformation

1. Thaw competent cells on ice.
2. Add DNA sample (1~20ul) to competent cells. leave them on ice for 30 minutes
3. Keep them in heating block for 45 seconds, then cool on ice for 2 minutes.
4. Add 40~200ul SOC liquid culture medium, then incubate under shaking culture for 1hour 37°C.
5. Seed it onto LB+antibiotics agar culture. Incubate for 24 hours at 37°C.

PCR

PCR was performed using Wizard® SV Gel and PCR, KAPA™HiFi HotStart ReadyMix (2x) according to the manufacturer's protocols

Westernblotting (Membrane fraction)

Sample Preparation

1. Cultivate 100ml of E. coli culture whose OD600 values are 0.5
2. Divide the 100ml cell culture in to 50ml tube, centrifuge 5000 rpm for 10 min
3. Wash with PBS 30ml, centrifuge 5000 rpm for 10 min
4. Resuspend with 50mM Tris(pH8.0) 100mM NaCl 10% glycerol 10ml
5. Sonicate both tubes
6. Centrifuge 4000 rpm for 10 min at room temperature
7. Ultracentrifuge the supernatant at 45000 rpm (around 190000g) at room temperature, then remove the resulting supernatant
8. Resuspend in guanidine buffer (50mM Tris (8.0) 300mM NaCl 10mM Imidazole 6M Guanidine-HCl 0.1% NP40 1mM DTT) to denature proteins
9. Use Ni-NTA beads to purify the target protein
10. Resuspend the purified protein in SDS-buffer
11. Follow the protocol for Westernblotting

以下：プライマーリスト

Sequence

We outsourced the sequencing to Macrogen
http://www.macrogen-japan.co.jp/cap_seq_0203.php