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<BIOMOD|2012|Titech Nano-Jugglers

Protocols

Vapor Deposition

Preparation for deposition

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

1st Deposition

increase the pressure in Bell jar.
put cover off and lay the jar.
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〗^(-3)Pa )

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

5. increase electric current slowly.

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

(Au): confirm melting at 8A and keep 11A 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×〖10〗^1)

※※

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

2nd Deposition

Repeat※※

Reagent

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

↑

EDAC conjugation

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

↑

Gold and thiol modified DNA conjugation

Weight 0.25 mg of gold-vapored 10 μm polystyrene beads into a 1.5 ml eppendorf tube.
Suspend micro-bieds in 1 ml of Milli-Q water
Pellet the micro-bieds via centrifugation for 15seconds at 1260-2680 xG
Remove platinum agglomerate
Centrifuge mixture for 10 minutes at 15000 xG
Resuspend micro-beads in 0.4 ml of 1M NaOH
Incubate for 1h at room temperature with gentle mixing
Centrifuge mixture for 10 minutes at 15000 xG
Resuspend micro-bieds pellet in 1 ml of Milli-Q water
Repeat steps 6-7 for five times
Resuspend micro-beads pellet in 20μl of 10μM　thiol modified DNA and 10mM pH8 Phosphate buffer
Incubate for 24h at room temperature with gentle mixing
Add 10μl of 1M NaCl, then Incubated at room temperature for 2hours
Repeat steps 13 for 3 times.
Centrifuge mixture for 10 minutes at 15000 xG
Resuspend micro-bieds pellet in 3×ssc buffer
Repeat steps 15-16 for 3 times.
Store particles at 4℃ in 3×ssc buffer

↑

Confirmation of Self assembled monolayer

Providing a cover glass with gold deposition
Immersed cover glass in 1M NaOH solution for 1 hour
Rinse NaOH with MilliQ water twice
Drip 5μl of 10μM　thiol modified DNA and 10mM pH8 Phosphate buffer
Incubated for 24 hours at room temperature
Add 2μl of 1M NaCl, then Incubated at room temperature for 2hours
Repeat steps 6 for 3 times.
Rinse with 3×ssc buffer twice
Drip 5μl of 10μM Fluorescent beads with a complementary DNA
Incubated for 20 minutes at room temperature
Observing the fluorescence

↑

Platinum particle and thiol –modified DNA conjugation

Weight 1 mg of 0.15-0.40 μm platinum particle into a 1.5 ml eppendorf tube.
Suspend micro-bieds in 1 ml of Milli-Q water
Pellet the micro-bieds via centrifugation for 15seconds at 1260-2680 xG
Remove platinum agglomerate
Centrifuge mixture for 10 minutes at 15000 xG
Resuspend micro-beads in 0.4 ml of 1M NaOH
Incubate for 1h at room temperature with gentle mixing
Centrifuge mixture for 10 minutes at 15000 xG
Resuspend micro-bieds pellet in 1 ml of Milli-Q water
Repeat steps 6-7 for five times
Resuspend micro-beads pellet in 20μl of 10μM　thiol modified DNA and 10mM pH8 Phosphate buffer
Incubate for 24h at room temperature with gentle mixing
Add 10μl of 1M NaCl, then Incubated at room temperature for 2hours
Repeat steps 13 for 3 times.
Centrifuge mixture for 10 minutes at 15000 xG
Resuspend micro-bieds pellet in 3×ssc buffer
Repeat steps 15-16 for 3 times.
Store particles at 4℃ in 3×ssc buffer

↑

Catalyst conjugation

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

↑

PAGE protocol

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

↑

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

make sample

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

40% acrylamide gel 5mL
10x TBE 1mL
MilliQ 4mL

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	18µL		16µL		8µL
10％H₂O₂	0µL	0%	2µL	1%	10µL	5%
100µM ssDNA F	0.5µL	2.5µM	0.5µL	2.5µM	0.5µL	2.5µM
100µM ssDNA R	0.5µL	2.5µM	0.5µL	2.5µM	0.5µL	2.5µM
10xSSC	1µL	0.5x	1µL	0.5x	1µL	0.5x
Mass	20 µL		20µL		20µL

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

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

3.anealing complementary strands for 10 minutes at 80℃

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

5. Load wells with sample solution 5ul.(sample 3ul + 2x BPB solution 3ul = sample solution 6ul)

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

7. Put the gels into a cyber gold solution taper.(SYBR-Gold solution: 10000x cyber gold 20ul + MilliQ water 200ml)

8. Shake the taper by hand for 10 minutes.(using used cyber 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.

↑

Observation of the platinum particles

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

↑

Observation of platinum hemisphere in solution of H2O2

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

↑

Observation of catalase conjugated 10 micro beads in solution of H2O2

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

↑

Observation of dissociation of dsDNA with UV-light

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

solution A

solution	amount	strength
100μM seqAazo4_5thiol	3μL	10μM
2xSSC	15μL	1x
MlliQ water	22μL
Mass	30μL

100μM seqAazo4_5thiol(DNAⅰ) 5μL
2xSSC 25μL
MlliQ water 20μL

solution B

solution	amount	strength
100μM seqAc_5thiol	3μL	10μM
2xSSC	155μL	1x
MlliQ water	12μL
Mass	30μL

100μM seqAc_5thiol(DNAⅱ) 5μl
2xSSC 25μL
MlliQ water 20μL

Solution A+B

solution	amount	strength
A	30μL	(DNAⅰ)5μM
B	30μL	(DNAⅱ)5μM
Mass	60μL

solution A 30μL
solution B 30μL

Finally prepared solution

solution	amount
Solution A	20μL
Solution B	20μL
Solution A+B	60μL

Way to hybridize DNA

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
mix 40 μl of each solution under the visible light
irradiate visible light for 10minutes again
Put solution into dark box at 4 ℃ for 12 hours to cool down for hybridization

Irradiate UV-light to DNA duplex and measures absorbance

Irradiated UV-light(365nm,30mW/㎠) to a solution A+B for 1, 5　minutes
Measured absorbance of each DNA solutions(1~5) near 260nm 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)

Irradiated UV-light(365nm,180mW/㎠) to a solution A+B for 5,10,30,40,50seconds.
Measured absorbance of each DNA solutions(1~8) near 260nm 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)

↑

Material Lists and Kits

Name	grade	Supplier	Product code	Cat No	Lot No
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, pH7.0	Biochmistry	Wako	013-23291	***	STF3372
Ultra PureTM 1M Tris-HCL pH 8.0	***	invitrogen^TM	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 20x Concentrate	***	SIGMA	***	S6639-1L	021M8403
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride	SU	TCI	D1601	25952-53-8	FJXDI
Suberic acid bis(3-sulfo-N-hydroxysuccinimide ester) sodium salt	***	SIGMA	S5799-25MG	***	051M4067V
SYBR Gold nucleic acid gel stain	***	life technologies^TM	S11494	***	927072
Urea	for Molecular Biology	Wako	211-01213	57-13-6	LAQ5967
Tris-Borate-EDTA Buffer (10X), 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