Biomod/2012/Titech/Nano-Jugglers/Protocols: Difference between revisions

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|MlliQ water
|MlliQ water
|20μL
|20μL
|-
|<span style="color:red">Mass</span>  
|<span style="color:red">Mass</span>  
|<span style="color:red">50μL</span>  
|<span style="color:red">50μL</span>  
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|
|
|}
|}
*100μM seqAc_5thiol(DNAⅱ)  5μl
*2xSSC  25μL
*MlliQ water  20μL
===Solution A+B===
:{| border="1"
|solution
|amount
|strength
|-
|A
|30μL
|(DNAⅰ)5μM
|-
|B
|30μL
|(DNAⅱ)5μM
|-
|Mass
|60μL
|
|}
*solution A 30μL
*solution B 30μL

Revision as of 07:34, 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

  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 + 1×TBE 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.

Thiol modified DNA and gold conjugation

  1. weight 0.25 mg of gold-vapored 10 μm polystyrene beads into a 1.5 ml eppendorf tube.
  2. Suspend micro-bieds in 1 ml of Milli-Q water
  3. Pellet the micro-bieds via centrifugation for 10minutes at 15000xG
  4. Resuspend micro-beads in 0.4 ml of 1M NaOH.
  5. Incubate for 1h at room temperature with gentle mixing.
  6. Centrifuge mixture for 10 minutes at 15000 xG
  7. Resuspend micro-bieds pellet in 1 ml of Milli-Q water
  8. Repeat steps 6-7 for five times.
  9. Resuspend micro-beads pellet in 45μl of 99.5% EtOH and 5μl　of 100 μM thiol-modified DNA
  10. Incubate for 24h at room temperature with gentle mixing.
  11. Centrifuge mixture for 10 minutes at 15000 xG
  12. Resuspend micro-bieds pellet in 99.5% EtOH
  13. Repeat steps 11-12 for 3 times.
  14. Store particles at 4℃ in 99.5% EtOH

Platinum particle and thiol –modified DNA conjugation

  1. weight 1 mg of 0.15-0.40 μm platinum particle into a 1.5 ml eppendorf tube.
  2. Suspend micro-bieds in 1 ml of Milli-Q water
  3. Pellet the micro-bieds via centrifugation for 15seconds at 1260-2680 xG
  4. Remove platinum agglomerate
  5. Centrifuge mixture for 10 minutes at 15000 xG
  6. Resuspend micro-beads in 0.4 ml of 1M NaOH.
  7. Incubate for 1h at room temperature with gentle mixing.
  8. Centrifuge mixture for 10 minutes at 15000 xG
  9. Resuspend micro-bieds pellet in 1 ml of Milli-Q water
  10. Repeat steps 6-7 for five times.
  11. Resuspend micro-beads pellet in 45μl of 99.5% EtOH and 5μl　of 100 μM thiol-modified DNA
  12. Incubate for 24h at room temperature with gentle mixing.
  13. Centrifuge mixture for 10 minutes at 15000 xG
  14. Resuspend micro-bieds pellet in 99.5% EtOH
  15. Repeat steps 13-14 for 3 times.
  16. Store particles at 4℃ in 99.5% EtOH


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.

Physical Vapor Deposition

  • Preparation for deposition
  1. pipette 10 μL 10μM polystyrene-beads into 2.0mL tube.
  2. add 200μL milliQ into the tube.
  3. mix the tube by vortex.
  4. extend the polystyrene-beads on the cover glass.
  5. dry the cover glass.
  • 1st Deposition
  1. increase the pressure in Bell jar.
  2. put cover off and lay the jar.
  3. 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)
※※
  1. wash the surface of the cover glass with 70% ethanol.
  2. wash the surface of the cover glass with 70% ethanol.
  3. pipette 70% ethanol and polystyrene-beads into a 2.0mL tube.
  4. centrifuge the tube by tabletop centrifuge.
  5. remove supernatant.
  6. repeat 3 to 5
  7. extend the polystyrene-beads over a cover glass.
  8. dry the cover glass.
  • 2nd Deposition
Repeat※※
  • Reagent
10μm polystyrene beads, Au 2cm, Cr, MilliQ, 70%ethanol

Azobenzen Protocol

Solution A

solution amount strength
100μM seqAazo4_5thiol 5μL 10μM
2xSSC 25μL 1x
MlliQ water 20μL
Mass 50μL
  • 100μM seqAazo4_5thiol(DNAⅰ) 5μL
  • 2xSSC 25μL
  • MlliQ water 20μL

solution B

solution amount strength
100μM seqAc_5thiol 5μL 10μM
2xSSC 25μL 1x
MlliQ water 20μL
Mass 50μL
  • 100μM seqAc_5thiol(DNAⅱ) 5μl
  • 2xSSC 25μL
  • MlliQ water 20μL


Solution A+B

solution amount strength
A 30μL (DNAⅰ)5μM
B 30μL (DNAⅱ)5μM
Mass 60μL
  • solution A 30μL
  • solution B 30μL
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