Difference between revisions of "Biomod/2012/UTokyo/UT-Hongo/Method"

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For fabricating microchannel with PDMS, firstly silicon wafer was coated by SU-8(Nippon Kayaku Co., Japan) using spin coating. Then, using mask which was patterned the designed channel, SU-8 was molded according to the channel shape by photolithography. After that, the mold was placed in a dish, and PDMS(SILPOT 184, Dow Coning) was poured on the mold. Dish was heated for 2 hours at 75  in a oven. After PDMS was cured by heating, PDMS was cut and arranged the shape. And then the bonding surface of PDMS block and glass was treated oxygen plasma (RIE-10NR, REACTIVE ION ETCHING SYSTEM, samco) for 5 seconds. Finally, PDMS block and glass were bonded not to take air in, and PDMS microchannel was obtained.
 
For fabricating microchannel with PDMS, firstly silicon wafer was coated by SU-8(Nippon Kayaku Co., Japan) using spin coating. Then, using mask which was patterned the designed channel, SU-8 was molded according to the channel shape by photolithography. After that, the mold was placed in a dish, and PDMS(SILPOT 184, Dow Coning) was poured on the mold. Dish was heated for 2 hours at 75  in a oven. After PDMS was cured by heating, PDMS was cut and arranged the shape. And then the bonding surface of PDMS block and glass was treated oxygen plasma (RIE-10NR, REACTIVE ION ETCHING SYSTEM, samco) for 5 seconds. Finally, PDMS block and glass were bonded not to take air in, and PDMS microchannel was obtained.
 
For the adsorption of DNA origami to clean or hydrophilic silicon dioxide surfaces, glass was cleaned with acetone and 2-propanol, then rinsed in DI water, assisted by ultrasonic agitation. Next, the surface was treated with oxygen plasma to yield a hydrophilic surface, using standard cleaning parameters (50% power and 0.5 mbar chamber pressure for 30 sec). A drop of 2  l 5ラ TAE buffer was dispensed onto the cleaned surface. The TAE buffer spreads completely over the cleaned surface. A 2  l drop of DNA origami solution was dispensed on top. The incubation time was 30 minutes in a closed petri dish. After that time the sample was dipped for 5 seconds into a solution of water and ethanol (50:50 v/v), followed by immersion for one hour in a solution of water in ethanol (10:90 v/v). All steps were done at room temperature.
 
  
 
=Reagent=
 
=Reagent=

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AFM

We used AFM (Atomic Force Microscopy) for observing shell shape which we made.

Principle

AFM is composed of cantilever, laser, photodiode, and detector and feedback electronics. AFM uses forces between the tip and sample. When cantilever approaches to sample, it deflected and this deflection is measured by laser. Reflected laser from cantilever is changed and its change is detected by photodiode and analyzed by feedback electronics

Scanning Mode

There are 3 kinds of scanning mode. In this project, we used trapping mode for getting shell images.

Trapping Mode

Move cantilever near to its resonance frequency, oscillate it up and down by piezoelectric. The amplitude of cantilever oscillation is decreased by interaction forces between cantilever and sample. Tapping mode lessens the damage to sample.

Static Mode

Cantilever drags the surface of sample. There are lots of noise and drift as dragging so low stiffness cantilevers are mainly used.

Non-contact Mode

Cantilever isn't contact with the sample. The distance between cantilever and sample is a few nanometers.

Photometer

Fluorophotometer

For observing the fluorescence of samples, fluorophotometer (F-2500, Hitachi High-Tech, Japan) was used. The process was following. Firstly, the machines were switched on in order of fluorophotometer, heat regulator, computer, monitor. Secondly, quartz cell and motor were washed by water, and dried. Then the sample and motor were put into the cell. The cell was set in the fluorophotometer and then the motor was switched on. After that, the fluorophotometer was run and the data was obtained.

Absorption Spectrophotometer

For quantifying DNA, the absorption spectrophotometer (TMSPC-8, SHIMADZU Co., Japan) was used. The process was following. Firstly, DNA solution was diluted in 10ラBuffer and pure water (Density after the dilution becomes the range of 5 ~ 125ng /?L). Secondly, 500?L 1ラBuffer was put into the cell and set in the photometer. Then the photometer was run and the reference data was obtained. After that, 500?L sample solution was put into the other cell, and repeat the same process.

Electrophoresis

Poly-Acrylamide Gel Electrophoresis

First, Prepare two glass plates, rubber and clips and wash the glass plates with ethylene alcohol. Fix them by clips. Next, pour 8.12ml of pure water, 3.13ml of 40% acrylamide and 1.25ml of 10ラTBE in a beaker. And add 125 l of 10% APS and 8 l of TEMD to the beaker and stir to mix for a few seconds. Then cast the gel into a mold and put it to the room temperature for around an hour. Then Put TBE buffer in a electrophoresis tank.Take off the clip and fix it with upper part laying upon, clip two of the tank. Electrophoresis at 200V in a state putting nothing in for around 30 minutes. After that Mix 6ラLoading Buffer(1 l) and each sample(5 l) and pour them into the gel. Electrophoresis at 200V for around 30 minutes. Then soak the gel in solution of the cyber gold for around 20 minutes. Finally, switch on a machine and attach a switch of the visible light. Put the gel on the machine, cut visible light and expose it to UV light. Set an exposure time and take a photo.

Agarose Gel Electrophoresis

Put 1ラTris-Borate-EDTA (KANTO CHEMICAL CO.,INC) buffer and agarose gel (Fast Gene) in beaker and dissolved it with microwave oven. Cast the gel into a mold and put it to the room temperature for around one hour. Next, set the gel at the device (Mupid-2plus, Takara). Mix 10ラLoading Buffer (Takara) and each sample and pour them into the gel. Switch on and start. Finally, Soak the gel in solution of the cyber gold (Takara) for around 30 minutes.

Ultraviolet Irradiation

Wear protective goggles and put a sample in a tube. Bring the tip of the irradiation machine on the tube Push Emission button and expose the tube to UV light for 60seconds.

Thermal Cycler

Switch on the Thermal cycler (Veriti?Thermal cycler, Applied Biosystems). Put regents in tubes and set them to the machine. Select a program and push RUN button. After the end, get the tubes out of the machine and switch off.

Microfluidics

For fabricating microchannel with PDMS, firstly silicon wafer was coated by SU-8(Nippon Kayaku Co., Japan) using spin coating. Then, using mask which was patterned the designed channel, SU-8 was molded according to the channel shape by photolithography. After that, the mold was placed in a dish, and PDMS(SILPOT 184, Dow Coning) was poured on the mold. Dish was heated for 2 hours at 75 in a oven. After PDMS was cured by heating, PDMS was cut and arranged the shape. And then the bonding surface of PDMS block and glass was treated oxygen plasma (RIE-10NR, REACTIVE ION ETCHING SYSTEM, samco) for 5 seconds. Finally, PDMS block and glass were bonded not to take air in, and PDMS microchannel was obtained.

Reagent

Name Company note
M13mp18 Single Strand DNA (Virion DNA) TAKARA BIO INC. product code: 3518
Streptavidin Promega Corporation product code: Z704A
staple DNA
Oligo@SIGMA-PCR SIGMA-ALDRICH JAPAN
Oligo@SIGMA-Plate SIGMA-ALDRICH JAPAN
Sodium acetate buffer solution, pH 5.2 ア 0.1 at 25 SIGMA-ALDRICH product code: S7899-100ML
UltraPure 1M Tris-HCL pH 8.0 invitrogen product code: 15668-025
Hydrogen Peroxide Wako product code: 081-04215
3, 3', 5, 5',-Tetramethylbenzidine Tokyo Chemical Ind. Co. product code: T1023


Reagent List
Name Selling agency Maker Grade Product code Lot number
M13mp18 Single Strand DNA (Virion DNA) TAKARA BIO INC. TAKARA BIOTECHNOLOGY (DALIAN) CO., LTD. 3518
Streptavidin Promega Corporation Z704A 20768
staple DNA
Oligo@SIGMA-PCR Sigma-Aldrich Japan
Oligo@SIGMA-Plate Sigma-Aldrich Japan
Sodium acetate buffer solution, pH 5.2 ア 0.1 at 25 SIGMA-ALDRICH S7899-100ML S7899-100ML
UltraPure 1M Tris-HCL pH 8.0 invitrogen 15668-025 1286308
Hydrogen Peroxide Wako special grade 081-04215
3, 3', 5, 5',-Tetramethylbenzidine TOKYO CHEMICAL INDUSTRY CO. TOKYO CHEMICAL INDUSTRY CO. T1023 GH01-CNBO


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   <h2 style="border-bottom: none;">BIOMOD 2012 Team UT-Hongo</h2>
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<h3><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo">Top</a></h3> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo#description">Abstract</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo#youtube">YouTube</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo#navi">Links</a></li> </ul>

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<h3><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Intro">Motives</a></h3> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Intro#Focus">Focus</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Intro#Idea_of_DNA_Shell">Idea</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Intro#Functionalities_Exhibited">Funcitonalities</a></li>

         <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Intro#Conceptual_Blueprint_of_the_Structure">Blueprint</a></li>

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<h3><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Assembly">Design & Results</a></h3> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Assembly#Design">Design</a></li>

         <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Assembly#Adding_functionality">Function</a></li>
         <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Assembly#Result">Result</a></li>
         <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Assembly#Assembly_of_the_DNA_Shell">Experiments</a></li>

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<h3><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Method">Method</a></h3> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Method#AFM">AFM</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Method#Photometer">Photometer</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Method#Electrophoresis">Electrophoresis</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Method#Ultraviolet_Irradiation">Others</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Method#Reagent">Reagent</a></li> </ul>

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<h3><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/FutureWork">Progress & Beyond</a></h3> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/FutureWork#Variety_of_Target_Substances">Target Variety</a></li>

         <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/FutureWork#DNA_Shell_with_Functionality">Functionalization</a></li>
         <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/FutureWork#Shell_with_the_DNA_Hybridization_Circuits">Circuits</a></li>
         <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/FutureWork#A_Device_more_than_Shell_and_Enzyme">Conclusion</a></li>

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<h3><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Team">Team</a></h3> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Team#Info">Info</a></li>

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<h3><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Acknowledgement">Acknowledgement</a></h3> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Acknowledgement#Mentor">Mentor</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Acknowledgement#Professors">Professors</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Acknowledgement#Sponsors">Sponsors</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Acknowledgement#Special_Thanks">Special Thanks</a></li> </ul>

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     Copyright (C) 2012 | Design by Yuichi Nishwiaki | BIOMOD Team UT-Hongo
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