Biomod/2015/OhioMOD/protocols

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<div id="title2"> Protocols</div> <div id="prestocks01copy"> <p>Pre-Stocks</p> <p> <ol type="1"> <li>Order oligonucleotides, “staples”, with programmed DNA sequences</li> <li>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. </li> <li>Centrifuge the 96 well plates for 2 minutes at 2000 rpm.</li> <li>Pipette the designated oligonucleotides from the oligonucleotide plate into the pre-stock plate.</li> <br></br> <br></br> <br></br> <br></br> <li>Mix the contents of each pre-stock well thoroughly with pipet.</li></p></div>

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<div id="prestocks02copy"> <p>Working Stocks</p> <p><ol type="1"> <li>Create working stock sheets outlining the pre-stock combinations to make a specific structure.</li> <li>Centrifuge pre-stock plates for 2 minutes at 2000 rpm.</li> <li>Label a 1.5 mL tube and pipette the required oligos as designated by the working stock sheet.</li> <li>Place the working stock tube on the vortexer for 5 seconds.</li> <li>Centrifuge the working stock tube for 10- 15 seconds.</li></ol></p>


<p><br>Folding Reactions</br></p> <p><ol type="1"></p> <li>Centrifuge working stock for 10-15 seconds.</li> <li>For a 20nM structure concentration in 50 uL volume with 10-fold excess staple concentration:</li> <p><ol type="a"> <li>Add 10 uL of 100 nM scaffold.</li> <li>Add 20 uL of 500 nM staples.</li> <li>Add 10 uL of dH20.</li> <li>Add 5 uL of 10x FOB buffer.</li> <li>Add 5 uL of 18mM MgCl2 salt.</li></ol></p> <li>Centrifuge for 10-15 seconds. </li> <li>Place in thermocycler for 4-hour time ramp at 53 degrees C. </li></p></ol>


<p><br>Agarose Gel Electrophoresis</br></p> <p>For 2% Agarose gels:</p> <p><br>Large Gel:</br></p> <p><ol type="1"> <li>Weigh 2.5 g Agarose in a beaker.</li> <li>Add 124 g of 0.5x TBE buffer.</li> <li>Microwave for 1-2 minutes to dissolve Agarose.</li> <li>Replace volume of evaporated water with ddH2O until weight returns to 124 g.</li> <li>Add 1 mL of 1.375 M MgCl2.</li> <li>Add 5 uL of Ethidium bromide intercalating dye.</li> <li>Swirl beaker to mix contents.</li> <li>Pour gel mixture into gel tray and insert comb.</li></ol></p>

<p><br>Small Gel:</br></p> <p><ol type="1"> <li>Weigh 1.0 g Agarose in a beaker.</li> <li>Add 49.6 g of 0.5x TBE buffer.</li> <li>Microwave for 1-2 minutes to dissolve Agarose.</li> <li>Replace volume of evaporated water with ddH2O until weight returns to 49.6 g.</li> <li>Add 0.4 mL of 1.375 M MgCl2.</li> <li>Add 2 uL of Ethidium bromide intercalating dye.</li> <li>Swirl beaker to mix contents.</li> <li>Pour gel mixture into gel tray and insert comb.</li></ol></p>


<p><br>Gel Loading:</br></p> <p><ol type="1"> <li>Mix 15 uL of sample with 3 uL of EDTA -free loading dye.</li> <li>For a 10nM scaffold solution, mix 1.5 uL of 100nM scaffold, 13.5 uL of dH20, and 3uL of EDTA-free loading dye.</li> <li>Once gel is solid, fill gel rig with TBE buffer containing MgCl2.</li> <li>Remove comb.</li> <li>Pipette 6uL of DNA ladder to the first gel well.</li> <li>Pipette 17 uL of prepared scaffold solution to the second gel well.</li> <li>Pipette 17 uL of desired sample to each well.</li> <li>Attach electrodes, connect to power supply, and run at 70 V.</li> <p><ol type="a"> <li>Small gels can be run for 1.5- 2 hours.</li> <li>Large gels can be run for 3-4 hours.</li></ol></p> <li>Pack ice around gel rig to avoid overheating of gel during electrophoresis.</li></ol></p>

<p><br>Ultraviolet Gel Imaging</br></p> <p><ol type="1"> <li>Place gel containing Ethidium Bromide intercalating dye on UV imaging table to show the location of the samples.</li> <li>Use camera to image the gel.</li> <p><ol type="a"> <li>The brightness, contrast, and focus may be adjusted to ensure clear images of gel bands.</li></ol></p></ol></p>


<p><br>TEM Grid Preparation</br></p> <p><ol type="1"> <li>Prepare Uranyl Formate (UFo) staining solution.</li> <p><ol type="a"> <li>Allow UFo to thaw in a foil- wrapped tube.</li> <li>Add 1.0 uL of 5.0 M NaOH to the side of the UFo tube to bring pH to 8.0.</li> <li>Vortex for 2 minutes.</li> <li>Centrifuge at 21,100 G for 3 minutes.</li> <li>Place tube back in foil wrapper to protect from light contamination.</li></ol></p> <li>Glow-discharge the carbon-coated TEM grids to create a hydrophilic surface.</li> <li>Apply 3.5 uL of sample solution onto the carbon-coated side of the grid.</li> <li>Allow the sample solution to be absorbed for 4 minutes.</li> <li>When sample solution has less than 1 minute of absorption left, pipette 10 uL and 20 uL of UFo staining solution onto Para film.</li> <li>Use filter paper to draw the sample solution off the edge of the grid.</li> <li>Collect the 10 uL droplet with the carbon-coated side of the grid and immediately remove with filter paper.</li> <li>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.</li> <li>Remove the 20 uL droplet with the filter paper.</li> <li>Place the finished grid into the grid box and allow to dry for at least 15 minutes.</li></ol></p>


<p><br>Gel Purification</br></p> <p><ol type="1"> <li>Cover the UV imaging table with a clear cutting board.</li> <li>Place gel containing Ethidium Bromide on the cutting board.</li> <li>Cut out the gel bands that display DNA fluorescence that may contain well folded structures.</li> <li>Place bands in a labeled crunch n’ squeeze tube.</li> <li>Centrifuge for 6 minutes at 13,000 rpm.</li> <li>Remove filter and store resulting filtrate with purified structures.</li></ol></p>


<p><br>PEG Purification</br></p> <p><ol type="1"> <li>In a 1.5 mL Eppendorf tube, add 150 uL of the desired structure.</li> <li>Add 150 uL of 15% PEG 8000.</li> <li>Vortex for 2 minutes.</li> <li>Centrifuge for 30 minutes at 16,000 rpm.</li> <li>Remove supernatant.</li> <li>Resuspend resulting pellet in desired buffer.</li></ol>



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