Biomod/2012/Titech/Nano-Jugglers/Protocols: Difference between revisions
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# Repeat Steps 12-13 for three times. | # Repeat Steps 12-13 for three times. | ||
# Store particles at 4˚C in Polylink Wash/Storage Buffer. | # Store particles at 4˚C in Polylink Wash/Storage Buffer. | ||
<h2>Physical Vapor Deposition</h2> | |||
*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 |
Revision as of 04:01, 2 September 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
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.
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.
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.
Physical 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