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Revision as of 18:00, 21 October 2012
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} </style> </head> <BODY> <div id="biomodlink"> <<a href="http://openwetware.org/wiki/Biomod">BIOMOD</a>|<a href="http://openwetware.org/wiki/Biomod/2012">2012</a>|Titech Nano-Jugglers </div> <div id="header"> <div id="navigation"> <div id="menu"> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers"><br>Home<br><br></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Team/Students"><br>Team<br><br></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Project"><br>Project<br><br></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Results">Results<br>&<br>Methods</a></font></li> <li class="ach"><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Achievements"><br>Achievements<br><br></a> <li class="sup"><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Protocols"><br>Suppl. Info.<br><br></a></li> <li class="none"><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Acknowledgement"><br>Acknowledgements<br><br></a></li> </ul> </div> </div> </div> </BODY> </html>
Protocols
Vapor Deposition
- Preparation for deposition
- pipette 10 μL 10μM polystyrene-beads into 2.0mL tube.
- add 200μL milliQ into the tube.
- mix the tube by vortex.
- extend the polystyrene-beads on the cover glass.
- dry the cover glass.
- 1st Deposition
- increase the pressure in Bell jar.
- put cover off and lay the jar.
- put target material (Cr, Au) in the basket.
- ※the amount of PVD is related to the Volume of targets.
- 4. vacuuming (to 1.0×〖10〗^(-3)Pa )
- <energization>
- ※Au can't connect with polystyrene beads, so we deposit Au after Cr.
- 5. increase electric current slowly.
- 6. (Cr) 1st: stop at 15A and keep 30 seconds. /2nd: stop at 15A and keep 50 seconds.
- (Au): confirm melting at 8A and keep 11A until all Au has evaporated.
- 7. off electric current
- 8. cool down
- 9. increase pressure
- 10. take out the sample
- 11. vacuuming(to 1.0×〖10〗^1)
- ※※
- wash the surface of the cover glass with 70% ethanol.
- wash the surface of the cover glass with 70% ethanol.
- pipette 70% ethanol and polystyrene-beads into a 2.0mL tube.
- centrifuge the tube by tabletop centrifuge.
- remove supernatant.
- repeat 3 to 5
- extend the polystyrene-beads over a cover glass.
- dry the cover glass.
- 2nd Deposition
- Repeat※※
- Reagent
- 10μm polystyrene beads, Au 2cm, Cr, MilliQ, 70%ethanol
EDAC conjugation
- Pipet 12.5mg of polystirene-beads into a 1.5mL tube.
- Pellet the micro-beads via centrifugation for 5 minutes at 1000 xG.
- Resuspend micro-beads pellet in 0.4 ml of PolyLink Coupling Buffer.
- Pellet again via centrifugation for 5 minutes at 1000 G.
- Resuspend the micro-beads pellet in 0.17 ml of PolyLink Coupling Buffer.
- Just before use, prepare a 200 mg/ml EDAC solution by dissolving 10mg PolyLink EDAC in 50µl Polylink Coupling Buffer.
- Add 20 µl of the EDAC solution to the micro-beads suspension.
- Mix gently end-over-end or briefly vortex.
- Add 5 nmol of aminated DNA. Mix gently end-over-end or briefly vortex.
- Incubate for 90 minutes at room temperature with gentle mixing.
- Centrifuge mixture for 10 minutes at 1000 x G.
- Resuspend micro-beads pellet in 0.4ml Polylink Wash/Storage Buffer.
- Repeat Steps 12-13 for three times.
- Store particles at 4˚C in Polylink Wash/Storage Buffer.
Confirmation of EDAC conjugation
- Pipet 12.5mg of EDAC conjugated polystirene-beads into a 1.5mL tube.
- Pellet the micro-beads via centrifugation for 5 minutes at 1000 xG.
- Resuspend micro-beads pellet in 0.1 ml of 3×SSC Buffer
- Drip 5μl of 10μM Fluorescent beads with a complementary DNA
- Incubated for 10 minutes at 90℃
- Incubated for 40 minutes at room temperature
- Observing the fluorescence
Observation Conditions
- ISO6400
- Exposure time(Transmitted Light)1/100 seconds
- Exposure time(Blue Light)2seconds
- Magnification 10×40=400
Gold and thiol modified DNA conjugation
- Weight 0.25 mg of gold-vapored 10 μm polystyrene beads into a 1.5 ml eppendorf tube.
- Suspend micro-bieds in 1 ml of Milli-Q water
- Pellet the micro-bieds via centrifugation for 15seconds at 1260-2680 xG
- Remove platinum agglomerate
- Centrifuge mixture for 10 minutes at 15000 xG
- Resuspend micro-beads in 0.4 ml of 1M NaOH
- Incubate for 1h at room temperature with gentle mixing
- Centrifuge mixture for 10 minutes at 15000 xG
- Resuspend micro-bieds pellet in 1 ml of Milli-Q water
- Repeat steps 6-7 for five times
- Resuspend micro-beads pellet in 20μl of 10μM thiol modified DNA and 10mM pH8 Phosphate buffer
- Incubate for 24h at room temperature with gentle mixing
- Add 10μl of 1M NaCl, then Incubated at room temperature for 2hours
- Repeat steps 13 for 3 times.
- Centrifuge mixture for 10 minutes at 15000 xG
- Resuspend micro-bieds pellet in 3×ssc buffer
- Repeat steps 15-16 for 3 times.
- Store particles at 4℃ in 3×ssc buffer
Platinum particle and thiol –modified DNA conjugation
- Weight 1 mg of 0.15-0.40 μm platinum particle into a 1.5 ml eppendorf tube.
- Suspend micro-bieds in 1 ml of Milli-Q water
- Pellet the micro-bieds via centrifugation for 15seconds at 1260-2680 xG
- Remove platinum agglomerate
- Centrifuge mixture for 10 minutes at 15000 xG
- Resuspend micro-beads in 0.4 ml of 1M NaOH
- Incubate for 1h at room temperature with gentle mixing
- Centrifuge mixture for 10 minutes at 15000 xG
- Resuspend micro-bieds pellet in 1 ml of Milli-Q water
- Repeat steps 6-7 for five times
- Resuspend micro-beads pellet in 20μl of 10μM thiol modified DNA and 10mM pH8 Phosphate buffer
- Incubate for 24h at room temperature with gentle mixing
- Add 10μl of 1M NaCl, then Incubated at room temperature for 2hours
- Repeat steps 13 for 3 times.
- Centrifuge mixture for 10 minutes at 15000 xG
- Resuspend micro-bieds pellet in 3×ssc buffer
- Repeat steps 15-16 for 3 times.
- Store particles at 4℃ in 3×ssc buffer
Catalyst conjugation
- Weight 1mg of thiolated DNA modified platinum particles and DNA modified polystyrene beads into a 1.5 ml eppendorf tube
- Suspend micro-beads and platinum particles in 1 ml of 3×SSC buffer
- Incubate for 10 minutes at 90 ℃
- Incubate for 40 minutes at room temperature
PAGE protocol
- Make 20% polyacrylamide gel.
- Put into a microwave oven and liquefy urea for 15 seconds.
- 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 becomes coagulate(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 + 1×TBE 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.
Ascertain that DNA form duplex in 1~5% H₂O₂ solution without degenerated by H₂O₂
- make sample
solution amount strength 40%acrylamide gel 5mL 20% 10x TBE 1mL 1x Milli-Q 4mL Mass 10 mL
- 40% acrylamide gel 5mL
- 10x TBE 1mL
- MilliQ 4mL
- Way to make samples for electrophoresis
Make solutions ①~⑦ as table below
No. ① ① ② ② ③ ③ solution amount strengthen amount strengthen amount strengthen Milli-Q water 18µL 16µL 8µL 10%H₂O₂ 0µL 0% 2µL 1% 10µL 5% 100µM ssDNA F 0.5µL 2.5µM 0.5µL 2.5µM 0.5µL 2.5µM 100µM ssDNA R 0.5µL 2.5µM 0.5µL 2.5µM 0.5µL 2.5µM 10xSSC 1µL 0.5x 1µL 0.5x 1µL 0.5x Mass 20 µL 20µL 20µL
No. ④ ⑤ ⑥ ⑦ solution amount strengthen amount strengthen amount strengthen amount strengthen Milli-Q water 18.5µL 8.5µL 18.5µL 8.5µL 10%H₂O₂ 0µL 0% 10µL 1% 0µL 5% 10 100µM ssDNA F 0.5µL 2.5µM 0.5µL 2.5µM 0µL 0µM 0µL 0µM 100µM ssDNA R 0µL 0µM 0µL 0µM 0.5µL 2.5µM 0.5µL 2.5µM 10xSSC 1µL 0.5x 1µL 0.5x 1µL 0.5x 1µL 0.5x Mass 20μL 20μL 20μL 20μL
- 2. Incubate for 90minutes at room temperature with gentle mixing.
- 3.anealing complementary strands for 10 minutes at 80℃
- 4. Incubate for 90minutes at room temperature with gentle mixing.
- 5. Load wells with sample solution 5ul.(sample 3ul + 2x BPB solution 3ul = sample solution 6ul)
- 6. Run at specified voltage (250V or 300V) for specified time (50 minutes)
- 7. Put the gels into a cyber gold solution taper.(SYBR-Gold solution: 10000x cyber gold 20ul + MilliQ water 200ml)
- 8. Shake the taper by hand for 10 minutes.(using used cyber gold solution, shake for 15 or 20 minutes)
- 9. Take out the gels.
- 10. Spot blue light to observe the gel.
- 11. Take pictures through an orange filter by camera.
Observation of the platinum particles
- Make a solvent of about 100μL in an Eppendorf tube.
- Put beads in another Eppendorf tube with a spatula.
- Remove the dust on the surface of silicon rubber with the like Scotch tape.
- Drill holes in the silicon rubber with a belt punch.
- Press the holed silicon rubber to the surface of a dish.
- Add the beads in the tube to the solvent.
- Shake the tube by voltex and set in the Ultrasound device.
- Inject the beads solution into the hole of silicon rubber.
- Set the dish to the observation units after down the objective lens to the bottom.
- Observe with the eyepiece.
Observation of platinum hemisphere in solution of H2O2
- Weight 0.5 mg of platinum hemisphere particles into a schale
- Suspend 1~3% H2O2 solution until liquid surface is flat
- Wait 1 minute in order to stabilize bubble emissions
- Observed with a microscope
Observation of catalase conjugated 10 micro beads in solution of H2O2
- Weight 0.5 mg of catalase conjugated 10 micro beads into a schale
- Suspend 1~3% H2O2 solution until liquid surface is flat
- Wait 1 minute in order to stabilize bubble emissions
- Observed with a microscope
Observation of dissociation of dsDNA with UV-light
prepare
- 100μM seqA 4Azo_5thiol←DNAi
- 100μM seqAc 4Azo_5thiol←DNAii
- 2×SSC
- MilliQ water
solution A solution amount strength 100μM seqA 4Azo_5thiol 5μL 10μM 2xSSC 25μL 1x MlliQ water 20μL Mass 50μL
solution B solution amount strength 100μM seqAc_5thiol 5μL 10μM 2xSSC 25μL 1x MlliQ water 20μL Mass 50μL
Solution A+B solution amount strength A 50μL DNAi 5μM B 50μL DNAii 5μM Mass 100μL
Finally prepared solution solution amount Solution A 10μL Solution B 10μL Solution A+B 80μL
Way to hybridize DNA
- Irradiate visible-light(blue-light λ>400 nm)to solution A and B for 5 minutes with Visible-light irradiation equipment(Epi-Green Slim pro S)to stabilize trans-azobenzene
- mix 40 μl of each solution under the visible light
- irradiate visible light for 10minutes again
- Put solution into dark box at 4 ℃ for 12 hours to cool down for hybridization
Irradiate UV-light to DNA duplex and measures absorbance
- Irradiated UV-light(365nm,30mW/㎠) to a solution A+B for 1, 5 minutes
- Measured absorbance of each DNA solutions(1~5) near 260nm wavelength of light. The Abs of each DNA solution we measured was as follows(table below).
No Solusion (time exposed UV-light(MINUTES)) 1 A(0) 2 B(0) 3 A+B(0) 4 A+B(1) 5 A+B(5)
- Irradiated UV-light(365nm,180mW/㎠) to a solution A+B for 5,10,30,40,50seconds.
- Measured absorbance of each DNA solutions(1~8) near 260nm wavelength of light. The Abs of each DNA solution we measured was as follows(table below).
No Solusion (time exposed UV-light(SECONDS)) 1 A(0) 2 B(0) 3 A+B(0) 4 A+B(5) 5 A+B(10) 6 A+B(20) 7 A+B(30) 8 A+B(40) 9 A+B(50)
Material Lists and Kits
Name |
grade | Supplier |
Product code |
Cat No | Lot No |
Hydrogen peroxide(30%) | Wako 1st Grade | Wako | 081-04215 | *** | TLM1384 |
POLY BEAD CARBOXYLATE 10.0micron microsperes | *** | Polyscience | 9003-53-6 | 18133 | 643610 |
Glass beads GBL-40 | *** | APPIE | 101021 | *** | JISZ890 |
Platinum 0.15~0.45 μm 99.9% | *** | ALDRICH | 1001302221 | 1310-73-2 | MKBH4608V |
Platinum 1 μm | *** | micromer | 01-83-103 | *** | 1551201-01 |
Catalase, from Bovine Liver | Wako 1st Grade | Wako | 039-12901 | EC1.11.1.6 | LAG1488 |
Sodium Dihidrogenphosphate Dihydrate | Wako 1st Grade | Wako | 192-02835 | *** | LAR4091 |
Disodium Hydrogenphosphate 12-Water | Wako 1st Grade | Wako | 196-02835 | *** | LAQ5931 |
6×Lording Buffer Double Dye | Wako 1st Grade | Wako | 313-90351 | *** | 02008D |
Sodium Chloride | Wako 1st Grade | Wako | 191-01665 | *** | DCN6646 |
Sodium Hydroxide | Wako 1st Grade | Wako | 198-13765 | 1310-73-2 | LAN1989 |
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 |
Ultra PureTM 1M Tris-HCL pH 8.0 | *** | invitrogenTM | 15568-025 | *** | 9949164 |
40(w/v)%-Acrylamide/Bis Mixed Solution (29:1) | SP | nacalai tesque | *** | *** | L1F7876 |
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 |
SYBR Gold nucleic acid gel stain | *** | life technologiesTM | S11494 | *** | 927072 |
Urea | for Molecular Biology | Wako | 211-01213 | 57-13-6 | LAQ5967 |
Tris-Borate-EDTA Buffer (10X), Nuclease an Protease tested [TBE Buffer] | *** | nacalai tesque | 35440-31 | *** | L1A6455 |
PolyLink - Protein Coupling Kit for COOH Microparticles For Microparticles 1.0 Micron or Larger | *** | Polysciences, Inc. | 24350 | *** | 631643 |
PolyLink EDAC | *** |
Polysciences, Inc. | 2435C | *** | 631321 |
Software
Sequence Design
- NUPACK: Software to design DNA arraignment.
- →Direct Link:http://www.nupack.org/partition/browser
- The DINAMelt Web Server: We used to know K_m of DNA strand.
- →Direct Link:http://mfold.rna.albany.edu/?q=DINAMelt
Software of editing
- Paint.net: Image processing software. We used it to control light and shade.
- →Direct Link:http://www.paint.net/
- Image J: Image analysis software. We used it to process photo of electrophoresis.
- →Direct Link:http://rsbweb.nih.gov/ij/
- Inkscape: We used it to draw figures.
- →Direct Link:http://inkscape.org/index.php?lang=en
- Chem Bio Draw: We used it mainly to draw chemical formula.