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<div id="navigation"> <div id="menu" style="position:static"> <ul> <li><a class="aMain" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo">Home</a></li> <li><a class="aTeam" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Team/Students">Team</a></li> <li><a class="aProject" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project">Project</a>  <li><font color="#ffffff">Results</font> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results">Experiments</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Simulations">Simulations</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Achievements/DNA_Devices">DNA Design</a></li> </ul></li>  <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Achievements">Achievements</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Future_works">Future works</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Notebook/Protocols">Protocols</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Notebook/Lab.notebook">Notes</a></li> <li><a class="aNotebook" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Sponsors/">Sponsors</a></li> <li><a class="aSitemap" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Sitemap">Sitemap</a></li>

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Protocols

PAGE protocol

Make 20% polyacrylamide gel.
Put into a microwave oven and liquefy urea.
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 harden (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 + MilliQ water 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.

Notes

How to make samples is here.

How to make BPB solution is here.

There are two type 20% polyacrilamide gels.

With urea type (Checking DNA ciliate)

40% acrylamide gel (acrylamide:bisacrylamide =29:1) 5ml

10x TBE 1ml

Urea 4.8g ( =4ml,8M )

Mass: about 10ml

Without urea type (Checking UV switch)

40% acrylamide gel (acrylamide:bisacrylamide =29:1) 5ml

10x TBE 1ml

MilliQ 4ml

Mass: about 10ml

How to make samples for electrophoresis

DNA ciliate

Make this solution A in the 0.6ml tube
50mM ZnSO₄ 2μL
5x SSC buffer 2ul
(To check the enzyme activity for deoxyribozyme, remember to make the following solution B because deoxyribozyme activity requires Zn²⁺.)
MilliQ water 2ul
5x SSC buffer 2ul
Prepare DNA ciliate solution and centrifuge (15000rpm, 1minute) to make precipitation (DNA ciliate solution)
Vacuum the precipitation 1.5~3.0 ul by pipette and put into the solutions (A and B)
Mass up solutions to 8.5 ul by MilliQ water
Pour 2uM substrate DNA 1.5ul into the solutions to start reaction
React for 10~15 hours
Stop deoxyribozyme reaction by 250mM EDTA 2.5ul to reaction solutions
Mix by vortex
Put the tube onto heating block (80°C) for 1 minute to separate substrate DNA from deoxyribozime DNA
Centrifuge (15000rpm,1minute) to make precipitation
Put the tube onto heating block (80°C) for 1 minute to separate substrate DNA from deoxyribozime DNA
Vacuum the supernatant 3ul by pipette and put into 0.6ul tubes for samples
Finish making samples(sample A is enzyme active sample, sample B is enzyme inactive sample)

negative control and positive control

Positive control

Making the following solution.
MilliQ water 3ul
5x SSC 2ul
Deoxyribozyme DNA 1.5ul
Substrate DNA 1.5ul
50mM ZnSO₄ 2ul
Mix by vortex
Vacuum 3ul by pipette and put into 0.6ul tubes for samples
Finish making positive control

Negative control

Making the following solution.
MilliQ water 5ul
5x SSC 2ul
Deoxyribozyme DNA 1.5ul
Substrate DNA 1.5ul
Mix by vortex
Vacuum 3ul by pipette and put into 0.6ul tubes for samples
Finish making negative control

How to make DNA ciliate by EDC and NHS

Put 200ul of 2.5% polystyrene beads solution(1)) into a 1.5ml tube.
Centrifuge the tube for 1minute at 15,000rpm, and then remove the supernatant.
Put 200ul PBS buffer (pH 8.0) into the tube, and disperse the beads by pipetting or vortex mixer.
Centrifuge the tube for 1minute at 15,000rpm, and then remove the supernatant.
Repeat 3, 4 for three times to displace the solvent to PBS buffer.
Put 800ul PBS buffer (pH 8.0) into the tube, and disperse the beads.
Put 200ul of 1M NHS solution(2)) into the tube, and mix with vortex mixer.
Put 0.0384g EDC-HCl into the tube, and mix with vortex mixer to dissolve.
React beads with NHS and EDC-HCl solution using vortex mixer for an hour.
Centrifuge the tube for 1minute at 15,000rpm, and then remove the supernatant.
Put 200ul PBS buffer (pH 8.0) into the tube and disperse the beads by pipetting or vortex mixer.
Centrifuge the tube for 1minute at 15,000rpm, and then remove the supernatant.
Repeat 11, 12 for three times(3)) to displace the solvent to PBS buffer.
Put 8ul of 50uM DNA solution(4)) into the tube, and mix by pipetting.
React beads with DNA solution using the micro tube rotator for more than 2 hours.
Centrifuge the tube for 1minute at 15,000rpm, and then remove the supernatant.
Put 200ul of 0.2% SDS solution into the tube, and mix by pipetting and vortex mixer.
Centrifuge the tube for 1minute at 15,000rpm, and then remove the supernatant.
Put 200ul of 0.1M Tris-HCl buffer into the tube, and disperse the beads by pipetting or vortex mixer.
Centrifuge the tube for 1minute at 15,000rpm, and then remove the supernatant.
Repeat 19, 20 for three times(3)) to displace the solvent to 0.1M Tris-HCl buffer.
Put 200ul of 0.1M Tris-HCl buffer into the tube, and mix with vortex mixer.
React beads with Tris-HCl buffer using the micro tube rotator for an hour.
Keep the tube in refrigerator (4°C)

Notes

(1)The density of polystyrene beads is about 1.1g/ml, so 200ul of 2.5% polystyrene beads solution contains about 5mg polystyrene beads.
(2)Prepare 1M NHS solution by dissolving 0.023g NHS in 200ul PBS buffer(pH 8.0).
(3)This process should be done quickly because NHS can be easily hydrolyzed.

How to make DNA ciliate by EDAC

We used "PolyLink Protein Coupling Kit for COOH Microspheres" in this protocol.

Pipet 12.5mg of micro-beads into a 1.5mL tube.
Pellet the micro-beads via centrifugation for 5-10 minutes at approximately 500-1000 xG.
Resuspend micro-beads pellet in 0.4ml of Polylink Coupling Buffer.
Pellet again via centrifugation for 5-10 minutes at approximately 500-1000 G.
Resuspend the micro-beads pellet in 0.17ml of Polylink Coupling Buffer.
Just before use, prepare a 200mg/ml EDAC solution by dissolving 10mg Polylink EDAC in 50µl Polylink Coupling Buffer.
Add 20µl of the EDAC solution to the micro-beads suspension.
Mix gently end-over-end or briefly vortex.
Add 5nmol of aminated DNA. Mix gently end-over-end or briefly vortex.
Incubate for 30-60 minutes at room temperature with gentle mixing.
Centrifuge mixture for 10 minutes at approximately 500-1000 x G.
Resuspend micro-beads pellet in 0.4ml Polylink Wash/Storage Buffer.
Repeat Steps 12-13.
Store particles at 4˚C in Polylink Wash/Storage Buffer.

UV switch protocol

UV switch protocol (To check by electrophoresis)

A…DNA Devices

B…block DNA

D…deoxyribozyme DNA

5x SSC…sodium citrate 75mM

This experiment should be in UV cut room to prevent isomerizes

Make DNA solutions in 5xSSC and 80mM MgCl₂ buffer.The density of DNA solutions is 0.9uM.How to make is written in #Note.
Mix A and B in 0.6 ml tubes to make solution A+B. Mix A and D in 0.6 ml tubes to make solution A+D. To check the A+D’s loss of color by UV, prepare two A+D solution’s tube
Solution A+B…A 0.3uM and B 0.6uM (Mix A and B)
Solution A+D…A 0.225uM and D 0.225uM (Mix A, D, and 80mM Mg²⁺ in 5x SSC solution)
(To make controls, don’t mix all solutions)
Open the tube caps and spot visible light (no UV) to the solutions for 30 minute.The distance from solutions to light is 9 cm.
Close the tube’s caps.
Turn on the heating blocks at 95°C.
Put tubes onto heating block for 2 minute. (don’t take out tubes from the heating block)
Turn off the heating block and wait until the heating block becomes room temperature. It takes about 2 hour.
Take out tubes from the heating block.
Open the tube’s caps and spot visible light (no UV) to the solutions for 30 min again.
Mix A + B and D in 0.6ul tube to make reaction solutions. (A 0.225uM, B 0.45uM, D 0.225uM) (To make controls, don’t mix all solutions.)
Close the tube’s caps.
Set an UV light (365nm) equipment and turn on. (To stabilize the light, wait for 3 min.)
Open tube’s caps and put tubes under UV light. The distance from solutions to UV light is 5 cm.
Turn off the room light.
Take out the tubes after spotting light for specific time and close the tube’s caps
Turn on the room light. The solutions are samples for electrophoresis.
Do electrophoresis. The protocol for electrophoresis is here

Note

DNA solutions (DNA’s density is 0.9uM)

100uM DNA (A or B or D) 1ul

1M MgCl₂ 9ul

10x SSC 56ul

MilliQ water 45ul

DNA’s solution in 5xSSC, Mg²⁺ 80mM 111ul

Control solutions for electrophoresis: 1. A 0.225uM; 2. B 0.450uM; 3. D 0.225uM; 4. A + B (A 0.225uM + B 0.450uM); 5. A + D (A 0.225uM + D 0.225uM); 6. A + D (spotted UV)…to check the UV-switching-trap-DNA’s loss of color by UV; 80mM Mg<spb>2+</spb> in 5x SSC solution is used for dilutions

DNA hybridization to the complementary DNA protcol

Pipette the hybridization mix (5×SSC, 0.2%SDS, 10uM DNA) on the spot DNA immobilized on glass. (Hybridization mix pipetted 20ul per coverslip.)
Carefully lay the coverslip face down on the top of the hybridization mix.
Place the glass and moist Kimwipes into the Petri dish. Seal up Petri dish by using Parafilm.
Hybridize for 1h.(45°C)
Cool Petri dish for 30 min at room temp.
Wash the glass into the 5×SSC buffer of about 5mm.
Wash the glass into the 1×SSC buffer of about 5mm twice.
Observe the glass filled 1×SSC buffer.

DNA hybridization to the beads immobilized complementary DNA protcol

Pipette the hybridization mix (5×SSC, 3%BSA and about 10uM beads immobilized DNA) on the spot DNA immobilized on glass. (Hybridization mix pipetted 20ul per coverslip.)
Carefully lay the coverslip face down on the top of the hybridization mix.
Place the glass and moist Kimwipes into the Petri dish. Seal up Petri dish by using Parafilm.
Hybridize for 1h.(45°C)
Cool Petri dish for 30 min at room temp.
Wash the glass into the hybridization buffer A (5×SSC, 3%BSA) of about 5mm.
Wash the glass into the hybridization buffer B (1×SSC, 3%BSA) of about 5mm twice.
Observe the glass filled hybridization buffer B (1×SSC, 3%BSA).

Micromachining for polyformaldehyde(acetal)-resin protocol

Cut polyacetal-resin at the size of slide with an electric saw.
Boot up the micro fine machining center.(Model Number: Roland MODELA MDX-40A)
Unlock the emergency button.
Push the power button.
Get the computer to start up which is attached to micro fine machining center.
Execute the software of ClickMill and Vpanel.(ClickMill is the software which designates micro-channel’s shape. And Vpanel is the software which set the position of the endmill. )
Fix polyacetal-resin on the base of micro fine machining center.
Attach the 1mm diameter endmill to micro fine machining center.
Set X, Y, and Z origins by using Vpanel.
Cut polyacetal-resin by using Vpanel if necessary.
Do face milling. (Do face milling means flatten the polyacetal-resin by endmill)
Change the endmill which adjusts to the micro-channel’s width.(If micro-channel’s width is more than 10um, you don’t need to change endmill. If micro-channel’s width is between 50 um and 100 um, you should change to the endmill which diameter is 0.5um. Don’t make micro channel whose width is less than 50 um because it is difficult to carve exactly.
Make micro-channel according to the drawing. The roughly shape of micro-channel is the Figure-1.
After carving, remove polyacetal-resin by using isopropyl alcohol (IPA).
Observe micro-channel by using an optical microscope.

PDMS Mold Preparation Protcol

Before you begin this experimentation, you must sterilize the experiment stand.
Prepare PDMS, curing agent, electronic weigh and plastic tube which volume is 50ml.
Determine 1/10th measurement of the amount of PDMS used and pipette curing agent to the plastic tube.
Mix PDMS and curing agent about 10 minutes.
Try not to have almost all bubbles in the PDMS by using vacuum desiccators on 30 minutes to one hour.
After you check no bubble, apply the PDMS to the sample mold. The sample mold is made by polyacetal.
Try not to have any bubbles in the PDMS by using vacuum desiccators on 30mimutes again.
Prepare to use heater. (Switch on a heater.)
Bake PDMS on the heater for 1 hour at 75°C.
After heating it, cool it to room temperature.
Remove fully cured PDMS samples from heater
Cut PDMS along micro channel.

Note

PDMS=Polydimethylsiloxane
PDMS and curing agent we used is Silpot 184

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
SILPOT 184	***	DOW CORNING TORAY	03255981	***	0006410158
Acetone	Wako 1st Grade	Wako	012-00343	67-64-1	DCN6798
2-Propanol	Wako 1st Grade	Wako	166-04831	67-63-0	DCM6848
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
Di(N-succinimidyl) Suberate	***	TCI	D3895	68528-80-3	CU3BM
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
Dimethyl Sulfoxide	Wako 1st Grade	Wako	043-07216	67-68-5	LAL4398
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
N-Hydroxysuccinimide	***	TCI	H0623	6066-82-6	EOC3E
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
Dimethyl Sulfoxide	for Molecular Biology	Wako	041-29351	67-68-5	DCN0023
Zinc Sulfate	Practical Grade	Wako	268-00422	7733-02-0	LAQ6174
Urea	for Molecular Biology	Wako	211-01213	57-13-6	LAQ5967
Magnesium Chloride	***	Wako	136-03995	7786-30-3	LAR4676
Tris-Borate-EDTA Buffer (10X), Nuclease an Protease tested [TBE Buffer]	***	nacalai tesque	35440-31	***	L1A6455
Ammonium Peroxodisulfate	Wako 1st Grade	Wako	018-03282	7727-54-0	HLH7635
N,N,N',N'-TETRAMETHYL- ETHYLENEDIAMINE	Wako 1st Grade	Wako	202-04003	110-18-9	EPF1167
0.5N EDTA (pH8.0)	***	Wako	311-90075	***	01481B
Glycerol	Wako 1st Grade	Wako	075-00616	56-81-5	LAQ5416
Disodium Hydrogenphosphate 12-Water	Wako 1st Grade	Wako	196-02835	10039-32-4	LAQ5931
Sodium Dihydrogenphosphate Dihydrate	Wako 1st Grade	Wako	192-02815	13472-35-0	LAR4091
Hydrochloride Acid	Wako 1st Grade	Wako	080-01066	7647-01-0	LAN1065
PolyLink - Protein Coupling Kit for COOH Microparticles For Microparticles 1.0 Micron or Larger	***	Polysciences, Inc.	24350	***	631643
PolyLink EDAC	***	Polysciences, Inc.	2435C	***	631321