Biomod/2012/UTokyo/UT-Hongo/Method: Difference between revisions

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{{Biomod/2012/UTokyo/UT-Hongo/Begin|pagetype=method|pagename=Method}}
{{Biomod/2012/UTokyo/UT-Hongo/Begin|pagetype=method|pagename=Method}}


=='''AFM'''==
=AFM=


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


==='''Principle'''===
==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
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.


==='''Scanning Mode'''===
==Scanning Mode==


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


===''Trapping Mode''===
===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.  
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''===
===Static Mode===


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


=='''Photometer'''==
=Photometer=
==='''Fluorophotometer'''===


F-2500形日立分光蛍光光度計
We used 2 kinds of Photometer, Fluorophotometer and Absorption Spectrophotometer.  
具体的な実験操作方法
1電源を入れる
1.1まずは測定器と温度調節器
1.2次にパソコンおよびモニタ


2セルの準備
==Fluorophotometer==
2.1石英セルとモーターがアルコールに浸っているチューブを持って流しに移動
2.2ピンセットを超純水で洗い、セルとモーターと取り出す
2.3セルとモーターを水で洗い、超純水を良くかける
(この後、セルは上部と底面以外触れない)
2.4エアダスターでセルを良く乾燥させる
(セルの内側にストローを触れないように入れて丁寧に
風圧でセルを落とさないように注意)


3観測
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.


3.1セルに最初の試料を加え、モーターを入れる
==Absorption Spectrophotometer==
3.2装置のふたを開け、セルをセットする
(このときセルの線が手前になるよう統一すると良い)
3.3モーターのスイッチを入れる
3.4ふたを閉め、画面で観測ボタンを選ぶ
(波長や出力はMethodで設定する)
3.5試料を追加する場合は、ふた上部のゴムをはがし行う
(ピペットの先端が下に到達するまでくわえない
出し切るまでピペットをひきあげない)
3.6観測が終わればSTOPを選択し、データを保存
(プロジェクトファイル、テキストファイル、画像ファイルの3種類が良い)


4後片付け
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.
4.1プログラムを終了する
(その際「ランプを消灯後終了」を選ぶ)
4.2セルとモーターは水で洗い、超純水を良くかけ、最初のチューブに戻す
4.3データはUSBメモリでコピー
4.4パソコンの電源を切る
4.5モーターのスイッチを切り、装置と温度調整器の電源を切る


=Electrophoresis=


==Poly-Acrylamide Gel Electrophoresis==


1. Switch on machines in order of fluorophotometer (F-2500, Hitachi High-Tech, Japan), heat regulator, computer, monitor.  
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.


2. Wash quartz cell and motor by water, and dry them.
==Agarose Gel Electrophoresis==


3. Put the sample and motor into the cell. Set the cell in the fluorophotometer and switch on the motor. Then, run the fluorophotometer and observe.  
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.


(4. After finish observing, wash the cell and motor again.)
=Ultraviolet Irradiation=
 
 
==='''Absorption Spectrophotometer'''===
 
Absorption spectrophotometer (TMSPC-8, SHIMADZU Co., Japan) was used for quantifying DNA.
 
TMSPC-8 Tm Analysis System  SHIMADZU CORPORATION
GeneQuant Pro
吸光度計は業者から送られてきたDNAを定量するために使用します。
使用前に、粉状のnmol単位のDNAは遠心分離器にかけて底に集め、記載されてい
る物質量を参考に超純水で100μMの溶液(DNA原液)を作っておきます。
 
①DNA原液をBufferで希釈
希釈後の濃度が5~125ng/μLの範囲で測定が可能なので、この範囲になるように調
節します。目安としてMWが10000ほどのオリゴヌクレオチドの場合は100倍(1μM)
に希釈するといいです。
DNA原液を50μL、10×Bufferを50μL、純水を400μL加えます（Bufferは用途に合わ
せてください)
 
②光度計の使用
本体後部の電源を入れ、Oligoを押します。ついでSet-upを押してコウロチョウを
10mmに、キシャクリツに数値を入力し、enterで変更を保存します。最初の画面
に戻ると1×Bufferを500μLセルに入れ、縦向きに光が当たるようセットします。set
refを押してリファレンス測定をします。
測定が終わると、サンプル溶液500μLを別のセルに入れセットし、Oligoまたは
enterボタンを押してサンプル測定をします。測定終了後表示された数値は希釈前
のDNA原液の濃度を示しています。３回ほど連続で測定し、平均値を参考数値に
するのがよいです。
使用後は電源を消しましょう。
 
1. Dilute a DNA solution in Buffer
Dilute DNA solution in 10×Buffer and pure water (Density after the dilution becomes the range of 5 ~ 125ng /μl).
 
2. Put 500µL 1X Buffer into the cell and set it in the photometer. Then run the photometer and observe as reference. After that, change the cell to the other with 500µL sample solution, and repeat the same process.
 
=='''Electrophoresis'''==
 
==='''Poly-Acrylamide Gel Electrophoresis'''===
 
1.ガラス板の組み立て
 
1.1ガラス板2種類を持ってくる
1.2泳動を行う面にエチルアルコールをかけ、キムワイプで拭く
1.3コの字型のゴムカバーとクリップ4つを持ってくる
1.4ゴムを乗せ、クリップで自立できるように固定
 
 
 
2アクリルアミドゲルの作成
2.1ビーカーとコームを持ってくる
2.2ビーカーにアクリルアミドゲルを作る
(10%の場合、超純水を8.12ml, 40%アクリルアミドを3.13ml, 10×TBEを1.25ml
その後、10%APSを125μl, TEMDを8μl、の二つをあらかじめピペットに準備
同時に入れ、すぐにビーカーを7秒くらい混ぜる
アクリルアミドは神経毒なので注意)
2.3ガラス板を傾け、ゲルを流し入れる
2.4コームを差し込む、泡が立たないように2回
2.5ガラス板を傾けても水面が傾かないくらい、ドライヤーを3～4分間あてる
2.6 30分くらい待つ
 
 
3泳動装置の準備
3.1泳動槽を2種類持ってくる
3.2泳動槽の下側（下）にTBEバッファーを入れる
3.3ガラス板からコームを抜き、コの字型のゴムをはがす
(クリップを一つはずす、ゴムをはがす、クリップを戻す、の繰り返し)
3.4クリップをはずし泳動槽の上側（上）に合わせ、クリップ2つで固定
3.5下に、泡が入らないように斜めに、上を差し込む
(泡が入った場合は、注射器で取り除く)
3.6 上にTBEバッファーをいれる
3.7注射器でレーンを1往復掃除
3.8電極をさし、何も入れていない状態で200Vの電圧を30分くらい加える
(通称プレラン、感電注意)
 
 
4,アプライ、泳動
4.1セロハンを一片切りとり、表面の保護紙をはがす
4.2セロハンの上に6×loading buffer(青)を1μlずつ20個配置
(作業が不安であれば10個配置して、アプライを繰り返す)
4.3サンプルを5μl取り、青の一滴と混ぜる
4.4混ぜた溶液を6μl取り、レーンに入れる
4.5 4.3から4.4をレーンの数だけ繰り返す
4.6電極をさし、200Vで30分くらい泳動する(感電注意)
 
 
5染色
 
5.1泳動槽を持って流しに移動
5.2バッファーを捨て、クリップを外し、ガラス板を取り外す
5.3ヘラでガラス板を分離する(この時ガラスを重ねないように注意)
5.4ゲルの表裏を示すため、スミを切っておく
5.5ビニール手袋をはめ、ヘラでゲルをガラス板からはがす
5.6サイバーゴールドの溶液に20分くらい浸す
(サイバーゴールドは発がん性物質なので超注意)
 
 
6観測
 
6.1撮影装置の電源を入れ、可視光のスイッチをつける
(UVのスイッチが切れていることも確認)
6.2手袋をしてヘラを使ってゲルを回収し、撮影装置の真ん中に乗せる
6.3装置の扉をしめ、可視光のスイッチを切り、UVのスイッチを入れる
(紫外光に注意、特に波長254nmの紫外光を使う場合は保護メガネをする)
6.4となりの装置の時間を示すボタンを押し、露光時間を決定する
6.5 SAVEを押して画像がコンパクトフラッシュに保存する
 
7後片付け(みんなで使う装置なので、手を抜かない)
7.1クリップ、泳動槽、ゴム、コームは水で洗い、超純水でかける
(クリップの閉じる部分、泳動槽のスミおよび配線、コームの隙間は念入りに)
7.2ビーカー、ガラス板は洗剤で洗い、最後に超純水をかける
(特に注ぎ口、ガラスのスミは念入りに)
7.3撮影装置はまずゲルのカスをさらい、燃えるゴミへ
7.4撮影装置の内側にエチルアルコールをふきかけ、キムワイプで念入りに拭く
7.5コンパクトフラッシュのデータをPCにコピー
 
 
1.Assembling of glass plates
 
Prepare two glass plates, rubber and clips and wash the glass plates with ethylene alcohol.
Fix them by clips.
 
2.Making of the acrylamide gel (10%)
 
Pour 8.12ml of pure water, 3.13ml of 40% acrylamide and 1.25ml of 10×TBE in a beaker.
Add 125μl of 10% APS and 8μl of TEMD to the beaker and stir to mix for a few seconds.
Cast the gel into a mold and put it to the room temperature for around an hour.
 
3.Prepare electrophoresis apparatus
 
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.
 
4.Electrophoresis
 
Mix 6×Loading Buffer(1μl) and each sample(5μl) and pour them into the gel.
Electrophoresis at 200V for around 30 minutes
 
5.Staining
 
Throw away the buffer, take off the clips and remove the glass plates.
Separate the glass plates and peel off the gel from the glass plates with a spatula.
Soak the gel in solution of the cyber gold for around 20 minutes.
 
6.Observance
 
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.
 
7.Cleanup
 
Wash clips, rubber, comb and electrophoresis tank with tap water and pour pure water.
Wash beaker and glass plates with detergent and pour pure water.
Wipe the inside of the machine with ethylene alcohol.
 
 
'''Agarose Gel Electrophoresis'''
 
1. Making an Agarose Gel
 
1.1 Put 1×Tris-Borate-EDTA (KANTO CHEMICAL CO.,INC) buffer and agarose gel (Fast Gene) in beaker and dissolved it with microwave oven.
 
1.2 Cast the gel into a mold and put it to the room temperature for around one hour.
 
2. Agarose Gel Electrophoresis
 
2.1 Set the gel at the device (Mupid-2plus, Takara).
 
2.2 Mix 10×Loading Buffer (Takara) and each sample and pour them into the gel.
 
2.3 Switch on and start.
 
3. Staining
Soak the gel in solution of the cyber gold (Takara) for around 30 minutes.
 
=='''Ultraviolet Irradiation'''==
1防護メガネをかける
 
2資料をサーマルサイクラー用の小さい8連チューブのひとつに入れる(ふたにシールを張って区別)
 
3照射装置の先端をチューブの上に持ってくる(AFMのライトで固定すると便利)
 
4 Emissionボタンを押し、100%で60秒間UV照射


Wear protective goggles and put a sample in a tube.
Wear protective goggles and put a sample in a tube.
Line 264: Line 63:
Push Emission button and expose the tube to UV light for 60seconds.
Push Emission button and expose the tube to UV light for 60seconds.


=='''Thermal Cycler'''==
=Thermal Cycler=
1サーマルサイクラーの電源を入れる
2上部のハンドルを立て、奥に押して開く
3 8連チューブ(切り離しても良い)に試料を入れ、ふたをしたものをセットする
4 USERを選択する
5温度スケジュールがなければEDITする
5 RUNを押し、温度スケジュールを選び、STARTする
6終わったら、取り出し、電源を切る


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


=='''Microfluidics'''==
=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.
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 μl 5× TAE buffer was dispensed onto the cleaned surface. The TAE buffer spreads completely over the cleaned surface. A 2 μl 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'''==


{| border="1" cellpadding="20" cellspacing="0"
{| border="1" cellpadding="20" cellspacing="0"
Line 302: Line 92:
| Oligo@SIGMA-Plate||SIGMA-ALDRICH JAPAN||
| Oligo@SIGMA-Plate||SIGMA-ALDRICH JAPAN||
|-
|-
| Sodium acetate buffer solution, pH 5.2 ± 0.1 at 25℃||SIGMA-ALDRICH||product code: S7899-100ML
| 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
| UltraPure 1M Tris-HCL pH 8.0||invitrogen||product code: 15668-025
Line 313: Line 103:


{| border="1" cellpadding="20" cellspacing="0"
{| border="1" cellpadding="20" cellspacing="0"
|+ 試薬一覧
|+ Reagent List
! style="border-style: solid; border-width: 1px"|名前
! style="border-style: solid; border-width: 1px"|Name
! style="border-style: solid; border-width: 1px"|販売元
! style="border-style: solid; border-width: 1px"|Selling agency
! style="border-style: solid; border-width: 1px"|製造元
! style="border-style: solid; border-width: 1px"|Maker
! style="border-style: solid; border-width: 1px"|グレード
! style="border-style: solid; border-width: 1px"|Grade
! style="border-style: solid; border-width: 1px"|製品コード
! style="border-style: solid; border-width: 1px"|Product code
! style="border-style: solid; border-width: 1px"|ロットナンバー
! style="border-style: solid; border-width: 1px"|Lot number
|-
|-
| M13mp18 Single Strand DNA (Virion DNA)||TAKARA BIO INC.||TAKARA BIOTECHNOLOGY (DALIAN) CO., LTD.|| ||3518||
| M13mp18 Single Strand DNA (Virion DNA)||TAKARA BIO INC.||TAKARA BIOTECHNOLOGY (DALIAN) CO., LTD.|| ||3518||
Line 327: Line 117:
| staple DNA|| || || || ||
| staple DNA|| || || || ||
|-
|-
| Oligo@SIGMA-PCR||シグマアルドリッチジャパン合同会社ライフサイエンス事業部|| || || ||
| Oligo@SIGMA-PCR||Sigma-Aldrich Japan || || || ||
|-
|-
| Oligo@SIGMA-Plate||シグマアルドリッチジャパン合同会社ライフサイエンス事業部|| || || ||
| Oligo@SIGMA-Plate||Sigma-Aldrich Japan|| || || ||
|-
|-
| Sodium acetate buffer solution, pH 5.2 ± 0.1 at 25℃||SIGMA-ALDRICH|| || ||S7899-100ML||S7899-100ML
| 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
| UltraPure 1M Tris-HCL pH 8.0||invitrogen|| || ||15668-025||1286308
|-
|-
| Hydrogen Peroxide||Wako|| ||特級||081-04215||  
| Hydrogen Peroxide||Wako|| ||special grade||081-04215||  
|-
|-
| 3, 3', 5, 5',-Tetramethylbenzidine||東京化成販売株式会社||東京化成工業株式会社|| ||T1023||GH01-CNBO
| 3, 3', 5, 5',-Tetramethylbenzidine||TOKYO CHEMICAL INDUSTRY CO.||TOKYO CHEMICAL INDUSTRY CO.|| ||T1023||GH01-CNBO
|}
|}
=='''Reagents'''==
'''1.Google Docs'''
'''2.試薬'''
試薬の調整
[[Image:Biomod-2012-UTokyo-UT-Hongo- | 350px]]




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{{Biomod/2012/UTokyo/UT-Hongo/End}}
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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 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.

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

We used 2 kinds of Photometer, Fluorophotometer and Absorption Spectrophotometer.

Fluorophotometer

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.

Absorption Spectrophotometer

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.

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
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 ± 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


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 ± 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


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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> 

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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>
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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> 

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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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