We used AFM (Atomic Force Microscopy) for observing shell structures.
AFM is composed of cantilever, laser, photodiode, detector and feedback electronics. AFM is scanning the sample by forces between the tip and the 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.
There are 3 kinds of scanning mode. In this project, we used trapping mode for getting shell images.
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.
Cantilever drags the surface of sample. There are lots of noise and drift as dragging so low stiffness cantilevers are mainly used.
Cantilever isn't contact with the sample. The distance between cantilever and sample is a few nanometers.
We used 2 kinds of Photometer, Fluorophotometer and Absorption Spectrophotometer.
For observing the fluorescence of samples, fluorophotometer (F-2500, Hitachi High-Tech, Japan) was used. The process is following. First, we turned on machines in order of fluorophotometer, heat regulator, computer, monitor. Second, washed quartz cell and motor by water and dried them. Then put the sample and motor into the cell. The cell was set in the fluorophotometer and then the motor was switched on. After that, we run fluorophotometer and obtain the the data.
For quantifying DNA, the absorption spectrophotometer (TMSPC-8, SHIMADZU Co., Japan) was used. The process is following. First, DNA solution was diluted by 10×Buffer and pure water (Density after the dilution is 5 ~ 125ng /μL). Second, we put 500μL 1×Buffer into the cell and set in the photometer. Then run the photometer and the reference data was obtained. After that, we put 500μL sample solution into the other cell, and repeat the same process.
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.
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.
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.
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.
|M13mp18 Single Strand DNA (Virion DNA)||TAKARA BIO INC.||product code: 3518|
|Streptavidin||Promega Corporation||product code: Z704A|
|Sodium acetate buffer solution, pH 5.2 ± 0.1 at 25°C||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|
|Name||Selling agency||Maker||Grade||Product code||Lot number|
|M13mp18 Single Strand DNA (Virion DNA)||TAKARA BIO INC.||TAKARA BIOTECHNOLOGY (DALIAN) CO., LTD.||3518|
|Sodium acetate buffer solution, pH 5.2 ± 0.1 at 25 °C||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|