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# Set the dish to the observation units after down the objective lens to the bottom.<br>
# Set the dish to the observation units after down the objective lens to the bottom.<br>
# Observe with the eyepiece.<br>
# Observe with the eyepiece.<br>
<h2>EDAC conjugation</h2>
# 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.

Revision as of 23:51, 31 August 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

  1. Make 20% polyacrylamide gel.
  2. Put into a microwave oven and liquefy urea for 15 seconds.
  3. Wait until the solution become the room temperature.
  4. Add 10% APS 150 ul and TEMED 4ul.
  5. Put the solution into the gel box.
  6. Insert comb and wait it becomes coagulate(about 30 minutes).
  7. Make another gel box and prepare double gel boxes.
  8. Set up a tub for electrophoresis.
  9. 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).
  10. Incline the tub to remove bubbles under gels.
  11. Clear wells of the gel by pipette to remove unharden gel solution.
  12. Connect the tub to a power source.
  13. Pre-run at specified voltage (250V or 300V) for 20 minutes.
  14. Clear wells of the gel by pipette again.
  15. Load wells with sample solution 5ul.(sample 3ul + 2x BPB solution 3ul = sample solution 6ul)
  16. Run at specified voltage (250V or 300V) for specified time (50 minutes)
  17. Put the gels into a cyber gold solution taper.(SYBR-Gold solution: 10000x cyber gold 20ul + MilliQ water 200ml)
  18. Shake the taper by hand for 10 minutes.(using used cyber gold solution, shake for 15 or 20 minutes)
  19. Take out the gels.
  20. Spot blue light to observe the gel.
  21. Take pictures through an orange filter by camera.

Observation of the platinum particles

  1. Make a solvent of about 100μL in an Eppendorf tube.
  2. Put beads in another Eppendorf tube with a spatula.
  3. Remove the dust on the surface of silicon rubber with the like Scotch tape.
  4. Drill holes in the silicon rubber with a belt punch.
  5. Press the holed silicon rubber to the surface of a dish.
  6. Add the beads in the tube to the solvent.
  7. Shake the tube by voltex and set in the Ultrasound device.
  8. Inject the beads solution into the hole of silicon rubber.
  9. Set the dish to the observation units after down the objective lens to the bottom.
  10. Observe with the eyepiece.

EDAC conjugation

  1. Pipet 12.5mg of polystirene-beads into a 1.5mL tube.
  2. Pellet the micro-beads via centrifugation for 5 minutes at 1000 xG.
  3. Resuspend micro-beads pellet in 0.4 ml of PolyLink Coupling Buffer.
  4. Pellet again via centrifugation for 5 minutes at 1000 G.
  5. Resuspend the micro-beads pellet in 0.17 ml of PolyLink Coupling Buffer.
  6. Just before use, prepare a 200 mg/ml EDAC solution by dissolving 10mg PolyLink EDAC in 50µl Polylink Coupling Buffer.
  7. Add 20 µl of the EDAC solution to the micro-beads suspension.
  8. Mix gently end-over-end or briefly vortex.
  9. Add 5 nmol of aminated DNA. Mix gently end-over-end or briefly vortex.
  10. Incubate for 90 minutes at room temperature with gentle mixing.
  11. Centrifuge mixture for 10 minutes at 1000 x G.
  12. Resuspend micro-beads pellet in 0.4ml Polylink Wash/Storage Buffer.
  13. Repeat Steps 12-13 for three times.
  14. Store particles at 4˚C in Polylink Wash/Storage Buffer.
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