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Protocols

    In this page, detailed protocols are described as supplementary information.

Contents 1 Protocols 1.1 Vapor Deposition 1.2 EDAC conjugation 1.3 Confirmation of EDAC conjugation 1.4 Gold and thiol modified DNA conjugation 1.5 Platinum particle and thiol –modified DNA conjugation 1.6 Catalyst conjugation 1.7 PAGE protocol 1.8 Ascertain that DNA form duplex in 1~5% H2O2 solution without degenerated by H2O2 1.9 Observation of the platinum particles 1.10 Observation of platinum hemisphere in solution of H2O2 1.11 Observation of catalase conjugated 10 micro beads in solution of H2O2 1.12 Observation of dissociation of dsDNA with UV-light 2 Material Lists and Kits 3 Software 3.1 Sequence Design 3.2 Software of editing 3.3 Software for YouTube

Vapor Deposition

• Preparation for deposition

1. pipette 10 µL 10 µM polystyrene-beads into 2.0 mL tube.
2. add 200 µL milliQ into the tube.
3. mix the tube by vortex.
4. extend the polystyrene-beads on the cover glass.
5. dry the cover glass.

• 1st Deposition

1. increase the pressure in Bell jar.
2. put cover off and lay the jar.
3. put target material (Cr, Au) in the basket.

※the amount of PVD is related to the Volume of targets.

4. vacuuming (to 1.0×10^-3Pa)

<energization>

※Au can't connect with polystyrene beads, so we deposit Au after Cr.

5. increase electric current slowly.

6. (Cr) 1st: stop at 15 A and keep 30 seconds. / 2nd: stop at 15 A and keep 50 seconds.

(Au): confirm melting at 8 A and keep 11 A until all Au has evaporated.

7. off electric current

8. cool down

9. increase pressure

10. take out the sample

11. vacuuming (to 1.0×10¹)

※※

1. wash the surface of the cover glass with 70% ethanol.
2. wash the surface of the cover glass with 70% ethanol.
3. pipette 70% ethanol and polystyrene-beads into a 2.0 mL tube.
4. centrifuge the tube by tabletop centrifuge.
5. remove supernatant.
6. repeat 3 to 5
7. extend the polystyrene-beads over a cover glass.
8. dry the cover glass.

• 2nd Deposition

Repeat※※

• Reagent

10 µm polystyrene beads, Au 2 cm, Cr, MilliQ, 70% ethanol

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EDAC conjugation

1. Pipet 12.5 mg of polystirene-beads into a 1.5 mL tube.
2. Pellet the micro-beads via centrifugation for 5 minutes at 1000×G.
3. Resuspend micro-beads pellet in 0.4 mL of PolyLink Coupling Buffer.
4. Pellet again via centrifugation for 5 minutes at 1000×G.
5. Resuspend the micro-beads pellet in 0.17 mL of PolyLink Coupling Buffer.
6. Just before use, prepare a 200 mg/mL EDAC solution by dissolving 10 mg PolyLink EDAC in 50 µL Polylink Coupling Buffer.
7. Add 20 µL of the EDAC solution to the micro-beads suspension.
8. Mix gently end-over-end or briefly vortex.
9. Add 5 nmol of aminated DNA. Mix gently end-over-end or briefly vortex.
10. Incubate for 90 minutes at room temperature with gentle mixing.
11. Centrifuge mixture for 10 minutes at 1000×G.
12. Resuspend micro-beads pellet in 0.4 mL Polylink Wash/Storage Buffer.
13. Repeat Steps 12-13 for three times.
14. Store particles at 4°C in Polylink Wash/Storage Buffer.

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Confirmation of EDAC conjugation

1. Pipet 12.5 mg of EDAC conjugated polystirene-beads into a 1.5 mL tube.
2. Pellet the micro-beads via centrifugation for 5 minutes at 1000×G.
3. Resuspend micro-beads pellet in 0.1 mL of 3×SSC Buffer
4. Drip 5 µL of 10 µM Fluorescent beads with a complementary DNA
5. Incubated for 10 minutes at 90°C
6. Incubated for 40 minutes at room temperature
7. Observing the fluorescence

Observation Conditions

ISO6400

Exposure time (Transmitted Light) 1/100 seconds

Exposure time (Blue Light) 2 seconds

Magnification 10×40=400

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Gold and thiol modified DNA conjugation

1. Weight 0.25 mg of gold-vapored 10 µm polystyrene beads into a 1.5 mL eppendorf tube.
2. Suspend micro-bieds in 1 mL of Milli-Q water
3. Pellet the micro-bieds via centrifugation for 15 seconds at 1260-2680×G
4. Remove platinum agglomerate
5. Centrifuge mixture for 10 minutes at 15000×G
6. Resuspend micro-beads in 0.4 mL of 1 M NaOH
7. Incubate for 1 hour at room temperature with gentle mixing
8. Centrifuge mixture for 10 minutes at 15000×G
9. Resuspend micro-bieds pellet in 1 mL of Milli-Q water
10. Repeat steps 6-7 for five times
11. Resuspend micro-beads pellet in 20 µL of 10 µM thiol modified DNA and 10 mM pH 8 Phosphate buffer
12. Incubate for 24 hours at room temperature with gentle mixing
13. Add 10 µL of 1 M NaCl, then Incubated at room temperature for 2 hours
14. Repeat steps 13 for 3 times.
15. Centrifuge mixture for 10 minutes at 15000×G
16. Resuspend micro-bieds pellet in 3×SSC buffer
17. Repeat steps 15-16 for 3 times.
18. Store particles at 4°C in 3×SSC buffer

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Platinum particle and thiol –modified DNA conjugation

1. Weight 1 mg of 0.15-0.40 µm platinum particle into a 1.5 mL eppendorf tube.
2. Suspend micro-bieds in 1 mL of Milli-Q water
3. Pellet the micro-bieds via centrifugation for 15 seconds at 1260-2680×G
4. Remove platinum agglomerate
5. Centrifuge mixture for 10 minutes at 15000×G
6. Resuspend micro-beads in 0.4 mL of 1 M NaOH
7. Incubate for 1 hour at room temperature with gentle mixing
8. Centrifuge mixture for 10 minutes at 15000×G
9. Resuspend micro-bieds pellet in 1 mL of Milli-Q water
10. Repeat steps 6-7 for five times
11. Resuspend micro-beads pellet in 20 µL of 10 µM thiol modified DNA and 10 mM pH 8 Phosphate buffer
12. Incubate for 24 hours at room temperature with gentle mixing
13. Add 10 µL of 1 M NaCl, then Incubated at room temperature for 2 hours
14. Repeat steps 13 for 3 times.
15. Centrifuge mixture for 10 minutes at 15000×G
16. Resuspend micro-bieds pellet in 3×SSC buffer
17. Repeat steps 15-16 for 3 times.
18. Store particles at 4°C in 3×SSC buffer

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Catalyst conjugation

1. Weight 1 mg of thiolated DNA modified platinum particles and DNA modified polystyrene beads into a 1.5 mL eppendorf tube
2. Suspend micro-beads and platinum particles in 1 mL of 3×SSC buffer
3. Incubate for 10 minutes at 90°C
4. Incubate for 40 minutes at room temperature

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PAGE protocol

1. Make 20% polyacrylamide gel.
   
2. Put into a microwave oven and liquefy urea for 15 seconds.
   
3. Wait until the solution become the room temperature.
   
4. Add 10% APS 150 µL and TEMED 4 µL.
   
5. Put the solution into the gel box.
   
6. Insert comb and wait it becomes coagulate (about 30 minutes).
   
7. Make another gel box and prepare double gel boxes.
   
8. Set up a tub for electrophoresis.
   
9. Set double gel boxes and pour 1×TBE running buffer into the tub (upper tub’s buffer is new running buffer, and lower tub’s buffer is recycle buffer).
   
10. Incline the tub to remove bubbles under gels.
    
11. Clear wells of the gel by pipette to remove unharden gel solution.
    
12. Connect the tub to a power source.
    
13. Pre-run at specified voltage (250 V or 300 V) for 20 minutes.
    
14. Clear wells of the gel by pipette again.
    
15. Load wells with sample solution 5 µL.(sample 3µL + 2×BPB solution 3 µL = sample solution 6 µL)
    
16. Run at specified voltage (250 V or 300 V) for specified time (50 minutes)
    
17. Put the gels into a SYBR gold solution taper. (SYBR-Gold solution: 10000×SYBR gold 20 µL + 1×TBE 200 mL)
    
18. Shake the taper by hand for 10 minutes. (using used SYBR gold solution, shake for 15 or 20 minutes)
    
19. Take out the gels.
    
20. Spot blue light to observe the gel.
    
21. Take pictures through an orange filter by camera.
    

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Ascertain that DNA form duplex in 1~5% H₂O₂ solution without degenerated by H₂O₂

1. make sample

solution	amount	strength
40% acrylamide gel	5 mL	20%
10×TBE	1 mL	1×
Milli-Q	4 mL
Mass	10 mL

• 40% acrylamide gel 5 mL
• 10×TBE 1 mL
• MilliQ 4 mL

• Way to make samples for electrophoresis
  

Make solutions ①～⑦ as table below

No.	①	①	②	②	③	③
solution	amount	strengthen	amount	strengthen	amount	strengthen
Milli-Q water	18 µL		16 µL		8 µL
10% H2O2	0 µL	0%	2 µL	1%	10 µL	5%
100 µM ssDNA F	0.5 µL	2.5 µM	0.5 µL	2.5 µM	0.5 µL	2.5 µM
100 µM ssDNA R	0.5 µL	2.5 µM	0.5 µL	2.5 µM	0.5 µL	2.5 µM
10×SSC	1 µL	0.5×	1 µL	0.5×	1 µL	0.5×
Mass	20 µL		20 µL		20 µL

No.	④		⑤		⑥		⑦
solution	amount	strengthen	amount	strengthen	amount	strengthen	amount	strengthen
Milli-Q water	18.5 µL		8.5 µL		18.5 µL		8.5 µL
10% H2O2	0 µL	0%	10 µL	1%	0 µL	5%	10
100 µM ssDNA F	0.5 µL	2.5 µM	0.5 µL	2.5 µM	0 µL	0 µM	0 µL	0 µM
100 µM ssDNA R	0 µL	0 µM	0 µL	0 µM	0.5 µL	2.5 µM	0.5 µL	2.5 µM
10×SSC	1 µL	0.5×	1 µL	0.5×	1 µL	0.5×	1 µL	0.5×
Mass	20 µL		20 µL		20 µL		20 µL

2. Incubate for 90 minutes at room temperature with gentle mixing.

3.anealing complementary strands for 10 minutes at 80°C

4. Incubate for 90 minutes at room temperature with gentle mixing.

5. Load wells with sample solution 5 µL. (sample 3 µL + 2×BPB solution 3 µL = sample solution 6 µL)

6. Run at specified voltage (250 V or 300 V) for specified time (50 minutes)

7. Put the gels into a SYBR gold solution taper. (SYBR-Gold solution: 10000×SYBR gold 20 µL + MilliQ water 200 mL)

8. Shake the taper by hand for 10 minutes.(using used SYBR gold solution, shake for 15 or 20 minutes)

9. Take out the gels.

10. Spot blue light to observe the gel.

11. Take pictures through an orange filter by camera.

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Observation of the platinum particles

1. Make a solvent of about 100 µL in an Eppendorf tube.
   
2. Put beads in another Eppendorf tube with a spatula.
   
3. Remove the dust on the surface of silicon rubber with the like Scotch tape.
   
4. Drill holes in the silicon rubber with a belt punch.
   
5. Press the holed silicon rubber to the surface of a dish.
   
6. Add the beads in the tube to the solvent.
   
7. Shake the tube by voltex and set in the Ultrasound device.
   
8. Inject the beads solution into the hole of silicon rubber.
   
9. Set the dish to the observation units after down the objective lens to the bottom.
   
10. Observe with the eyepiece.
    

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Observation of platinum hemisphere in solution of H₂O₂

1. Weight 0.5 mg of platinum hemisphere particles into a schale
2. Suspend 1~3% H₂O₂ solution until liquid surface is flat
3. Wait 1 minute in order to stabilize bubble emissions
4. Observed with a microscope

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Observation of catalase conjugated 10 micro beads in solution of H₂O₂

1. Weight 0.5 mg of catalase conjugated 10 micro beads into a schale
2. Suspend 1~3% H₂O₂ solution until liquid surface is flat
3. Wait 1 minute in order to stabilize bubble emissions
4. Observed with a microscope

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Observation of dissociation of dsDNA with UV-light

prepare

• 100 µM seqA 4Azo_5thiol←DNAi
• 100 µM seqAc 4Azo_5thiol←DNAii
• 2×SSC
• MilliQ water

solution A
solution	amount	strength
100 µM seqA 4Azo_5thiol	5 µL	10 µM
2×SSC	25 µL	1×
MlliQ water	20 µL
Mass	50 µL

solution B
solution	amount	strength
100 µM seqAc_5thiol	5 µL	10 µM
2×SSC	25 μL	1×
MlliQ water	20 µL
Mass	50 µL

Solution A+B
solution	amount	strength
A	50 µL	DNAi 5 µM
B	50 µL	DNAii 5 µM
Mass	100 µL

Finally prepared solution
solution	amount
Solution A	10 µL
Solution B	10 µL
Solution A+B	80 µL

Way to hybridize DNA

1. Irradiate visible-light (blue-light λ > 400 nm) to solution A and B for 5 minutes with Visible-light irradiation equipment (Epi-Green Slim pro S) to stabilize trans-azobenzene
2. mix 40 µL of each solution under the visible light
3. irradiate visible light for 10 minutes again
4. Put solution into dark box at 4°C for 12 hours to cool down for hybridization

Irradiate UV-light to DNA duplex and measures absorbance

1. Irradiated UV-light (365 nm, 30 mW/cm²) to a solution A+B for 1, 5 minutes
2. Measured absorbance of each DNA solutions (1~5) near 260 nm wavelength of light. The Abs of each DNA solution we measured was as follows (table below).

No	Solusion (time exposed UV-light (MINUTES) )
1	A (0)
2	B (0)
3	A+B (0)
4	A+B (1)
5	A+B (5)

1. Irradiated UV-light (365 nm, 180mW/cm²) to a solution A+B for 5, 10, 30, 40, 50 seconds.
2. Measured absorbance of each DNA solutions (1~8) near 260 nm wavelength of light. The Abs of each DNA solution we measured was as follows (table below).

No	Solusion (time exposed UV-light (SECONDS) )
1	A (0)
2	B (0)
3	A+B (0)
4	A+B (5)
5	A+B (10)
6	A+B (20)
7	A+B (30)
8	A+B (40)
9	A+B (50)

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Material Lists and Kits

Name	grade	Supplier	Product code	Cat No	Lot No
Hydrogen peroxide (30%)	Wako 1st Grade	Wako	081-04215	***	TLM1384
POLY BEAD CARBOXYLATE 10.0 micron microsperes	***	Polyscience	9003-53-6	18133	643610
Glass beads GBL-40	***	APPIE	101021	***	JISZ890
Platinum 0.15~0.45 µm 99.9%	***	ALDRICH	1001302221	1310-73-2	MKBH4608V
Platinum 1 µm	***	micromer	01-83-103	***	1551201-01
Catalase, from Bovine Liver	Wako 1st Grade	Wako	039-12901	EC1.11.1.6	LAG1488
Sodium Dihidrogenphosphate Dihydrate	Wako 1st Grade	Wako	192-02835	***	LAR4091
Disodium Hydrogenphosphate 12-Water	Wako 1st Grade	Wako	196-02835	***	LAQ5931
6×Lording Buffer Double Dye	Wako 1st Grade	Wako	313-90351	***	02008D
Sodium Chloride	Wako 1st Grade	Wako	191-01665	***	DCN6646
Sodium Hydroxide	Wako 1st Grade	Wako	198-13765	1310-73-2	LAN1989
Ethnol(99.5)	Wako 1st Grade	Wako	057-00451	64-17-5	DBM6540
Albumin, from Bovine Serum, Cohn Fraction V, pH 7.0	Biochmistry	Wako	013-23291	***	STF3372
Ultra PureTM 1 M Tris-HCL pH 8.0	***	invitrogenTM	15568-025	***	9949164
40 (w/v) %-Acrylamide/Bis Mixed Solution (29:1)	SP	nacalai tesque	***	***	L1F7876
Sodium Dodecyl Sulfate (SDS)	Wako 1st Grade	Wako	196-08675	151-4-3	LAN1411
SSC Buffer 20×Concentrate	***	SIGMA	***	S6639-1L	021M8403
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride	SU	TCI	D1601	25952-53-8	FJXDI
SYBR Gold nucleic acid gel stain	***	life technologiesTM	S11494	***	927072
Urea	for Molecular Biology	Wako	211-01213	57-13-6	LAQ5967
Tris-Borate-EDTA Buffer (10×), Nuclease an Protease tested [TBE Buffer]	***	nacalai tesque	35440-31	***	L1A6455
PolyLink - Protein Coupling Kit for COOH Microparticles For Microparticles 1.0 Micron or Larger	***	Polysciences, Inc.	24350	***	631643
PolyLink EDAC	***	Polysciences, Inc.	2435C	***	631321

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Software

Sequence Design

• NUPACK: Software to design DNA arraignment.

→Direct Link:http://www.nupack.org/partition/browser

• The DINAMelt Web Server: We used to know K_m of DNA strand.

→Direct Link:http://mfold.rna.albany.edu/?q=DINAMelt

Software of editing

• Paint.net: Image processing software. We used it to control light and shade.

→Direct Link:http://www.paint.net/

• Image J: Image analysis software. We used it to process photo of electrophoresis.

→Direct Link:http://rsbweb.nih.gov/ij/

• Inkscape: We used it to draw figures.

→Direct Link:http://inkscape.org/index.php?lang=en

• Chem Bio Draw: We used it mainly to draw chemical formula.

→Direct Link:http://www.cambridgesoft.com/software/details/?fid=15&pid=226

Software for YouTube

• Sakura editor: We used it to make music.

→Direct Link:http://oto.chu.jp/

• Sound Engine: We used it to edit music.

→Direct Link:http://soundengine.jp/

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