Biomod/2012/UTokyo/UT-Hongo/Method

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

Revision as of 03:46, 28 October 2012

The University of Tokyo

Contents

AFM

We used AFM (Atomic Force Microscopy) for observing shell structures.

Principle

AFM is composed of cantilever, laser, photodiode, detector and feedback electronics. AFM is scanning the sample by forces between the tip and sample. When cantilever approaches to the 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×Buffer and pure water (Density after the dilution becomes the range of 5 ~ 125ng /μL). Secondly, 500μL 1×Buffer was put into the cell and set in the photometer. Then the photometer was run and the reference data was obtained. After that, 500μL 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 10xTBE in a beaker. And add 125 μL of 10% APS and 8 μL 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 6xLoading Buffer(1 μL) and each sample(5 μL) 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 1xTris-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 10xLoading 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
StreptavidinPromega Corporationproduct code: Z704A
staple DNA
Oligo@SIGMA-PCRSIGMA-ALDRICH JAPAN
Oligo@SIGMA-PlateSIGMA-ALDRICH JAPAN
Sodium acetate buffer solution, pH 5.2 ± 0.1 at 25°C SIGMA-ALDRICHproduct code: S7899-100ML
UltraPure 1M Tris-HCL pH 8.0invitrogenproduct code: 15668-025
Hydrogen PeroxideWakoproduct code: 081-04215
3, 3', 5, 5',-TetramethylbenzidineTokyo 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
StreptavidinPromega Corporation Z704A20768
staple DNA
Oligo@SIGMA-PCRSigma-Aldrich Japan
Oligo@SIGMA-PlateSigma-Aldrich Japan
Sodium acetate buffer solution, pH 5.2 ± 0.1 at 25 °CSIGMA-ALDRICH S7899-100MLS7899-100ML
UltraPure 1M Tris-HCL pH 8.0invitrogen 15668-0251286308
Hydrogen PeroxideWako special grade081-04215
3, 3', 5, 5',-TetramethylbenzidineTOKYO CHEMICAL INDUSTRY CO.TOKYO CHEMICAL INDUSTRY CO. T1023GH01-CNBO


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