Biomod/2015/OhioMOD/protocols

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<a href="http://openwetware.org/wiki/Biomod/2015/OhioMOD"></a> <a href="http://openwetware.org/wiki/Biomod/2015/OhioMOD/labbook"></a> <a href="http://openwetware.org/wiki/Biomod/2015/OhioMOD/background"></a> <a href="http://openwetware.org/wiki/Biomod/2015/OhioMOD/team"></a> <a href="http://openwetware.org/wiki/Biomod/2015/OhioMOD/sponsors2"></a> <a href="http://openwetware.org/wiki/Biomod/2015/OhioMOD/structure"></a> <a href="http://openwetware.org/wiki/Biomod/2015/OhioMOD/video"></a>
LAB BOOK




<a href="http://openwetware.org/wiki/Biomod/2015/OhioMOD/results"></a>


<a href="http://openwetware.org/wiki/Biomod/2015/OhioMOD/schedule"></a> <a href="http://openwetware.org/wiki/Biomod/2015/OhioMOD/protocols"></a>
Protocols

Pre-Stocks

  1. Order oligonucleotides, “staples”, with programmed DNA sequences
  2. Based on the structure design, determine which staples should be combined into one pre-stock. Each pre-stock is to be a combination of staples of a specific part of the DNA origami structure. Create pre-stock sheets to document which staples go into which pre-stock.
  3. Centrifuge the 96 well plates for 2 minutes at 2000 rpm.
  4. Pipette the designated oligonucleotides from the oligonucleotide plate into the pre-stock plate.








  5. Mix the contents of each pre-stock well thoroughly with pipet.

Working Stocks

  1. Create working stock sheets outlining the pre-stock combinations to make a specific structure.
  2. Centrifuge pre-stock plates for 2 minutes at 2000 rpm.
  3. Label a 1.5 mL tube and pipette the required oligos as designated by the working stock sheet.
  4. Place the working stock tube on the vortexer for 5 seconds.
  5. Centrifuge the working stock tube for 10- 15 seconds.



Folding Reactions

  1. Centrifuge working stock for 10-15 seconds.
  2. For a 20nM structure concentration in 50 uL volume with 10-fold excess staple concentration:

    1. Add 10 uL of 100 nM scaffold.
    2. Add 20 uL of 500 nM staples.
    3. Add 10 uL of dH20.
    4. Add 5 uL of 10x FOB buffer.
    5. Add 5 uL of 18mM MgCl2 salt.

  3. Centrifuge for 10-15 seconds.
  4. Place in thermocycler for 4-hour time ramp at 53 degrees C.



Agarose Gel Electrophoresis

For 2% Agarose gels:


Large Gel:

  1. Weigh 2.5 g Agarose in a beaker.
  2. Add 124 g of 0.5x TBE buffer.
  3. Microwave for 1-2 minutes to dissolve Agarose.
  4. Replace volume of evaporated water with ddH2O until weight returns to 124 g.
  5. Add 1 mL of 1.375 M MgCl2.
  6. Add 5 uL of Ethidium bromide intercalating dye.
  7. Swirl beaker to mix contents.
  8. Pour gel mixture into gel tray and insert comb.


Small Gel:

  1. Weigh 1.0 g Agarose in a beaker.
  2. Add 49.6 g of 0.5x TBE buffer.
  3. Microwave for 1-2 minutes to dissolve Agarose.
  4. Replace volume of evaporated water with ddH2O until weight returns to 49.6 g.
  5. Add 0.4 mL of 1.375 M MgCl2.
  6. Add 2 uL of Ethidium bromide intercalating dye.
  7. Swirl beaker to mix contents.
  8. Pour gel mixture into gel tray and insert comb.



Gel Loading:

  1. Mix 15 uL of sample with 3 uL of EDTA -free loading dye.
  2. For a 10nM scaffold solution, mix 1.5 uL of 100nM scaffold, 13.5 uL of dH20, and 3uL of EDTA-free loading dye.
  3. Once gel is solid, fill gel rig with TBE buffer containing MgCl2.
  4. Remove comb.
  5. Pipette 6uL of DNA ladder to the first gel well.
  6. Pipette 17 uL of prepared scaffold solution to the second gel well.
  7. Pipette 17 uL of desired sample to each well.
  8. Attach electrodes, connect to power supply, and run at 70 V.

    1. Small gels can be run for 1.5- 2 hours.
    2. Large gels can be run for 3-4 hours.

  9. Pack ice around gel rig to avoid overheating of gel during electrophoresis.


Ultraviolet Gel Imaging

  1. Place gel containing Ethidium Bromide intercalating dye on UV imaging table to show the location of the samples.
  2. Use camera to image the gel.

    1. The brightness, contrast, and focus may be adjusted to ensure clear images of gel bands.



TEM Grid Preparation

  1. Prepare Uranyl Formate (UFo) staining solution.

    1. Allow UFo to thaw in a foil- wrapped tube.
    2. Add 1.0 uL of 5.0 M NaOH to the side of the UFo tube to bring pH to 8.0.
    3. Vortex for 2 minutes.
    4. Centrifuge at 21,100 G for 3 minutes.
    5. Place tube back in foil wrapper to protect from light contamination.

  2. Glow-discharge the carbon-coated TEM grids to create a hydrophilic surface.
  3. Apply 3.5 uL of sample solution onto the carbon-coated side of the grid.
  4. Allow the sample solution to be absorbed for 4 minutes.
  5. When sample solution has less than 1 minute of absorption left, pipette 10 uL and 20 uL of UFo staining solution onto Para film.
  6. Use filter paper to draw the sample solution off the edge of the grid.
  7. Collect the 10 uL droplet with the carbon-coated side of the grid and immediately remove with filter paper.
  8. Collect the 20 uL droplet with the carbon-coated side of the grid and allow to sit for 20 seconds to absorb the staining solution.
  9. Remove the 20 uL droplet with the filter paper.
  10. Place the finished grid into the grid box and allow to dry for at least 15 minutes.



Gel Purification

  1. Cover the UV imaging table with a clear cutting board.
  2. Place gel containing Ethidium Bromide on the cutting board.
  3. Cut out the gel bands that display DNA fluorescence that may contain well folded structures.
  4. Place bands in a labeled crunch n’ squeeze tube.
  5. Centrifuge for 6 minutes at 13,000 rpm.
  6. Remove filter and store resulting filtrate with purified structures.



PEG Purification

  1. In a 1.5 mL Eppendorf tube, add 150 uL of the desired structure.
  2. Add 150 uL of 15% PEG 8000.
  3. Vortex for 2 minutes.
  4. Centrifuge for 30 minutes at 16,000 rpm.
  5. Remove supernatant.
  6. Resuspend resulting pellet in desired buffer.



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