Biomod/2014/OhioMOD/protocols: Difference between revisions

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<div id="protocols"> <h1>Lab Protocols</h1>

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     <TH COLSPAN="2"><strong>Table of Contents</strong>
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<A HREF="#scroll1">1. DNA origami structure production </br><ul id="list2"><A HREF="#scroll1.1">1.1. Pre-stocks</ul> <ul id="list2"><A HREF="#scroll1.2">1.2. Working stocks</ul> <ul id="list2"><A HREF="#scroll1.3">1.3. Folding reactions</ul> <A HREF="#scroll2">2. Imaging <ul id="list2"><A HREF="#scroll2.1">2.1. Agarose gel electrophoresis <ul id="list2"><A HREF="#scroll2.1.1">2.1.1. UV imaging</ul> <ul id="list2"><A HREF="#scroll2.1.2">2.1.2. Typhoon imaging</ul></ul> <ul id="list2"><A HREF="#scroll2.2">2.2. Sample preparation for TEM</ul> <ul id="list2"><A HREF="#scroll2.3">2.3. Sample preparation for TIRF</ul> <A HREF="#scroll3">3. Purification </br><ul id="list2"><A HREF="#scroll3.1">3.1. Agarose gel electrophoresis</ul> <ul id="list2"><A HREF="#scroll3.2">3.2. PEG purification</ul> <A HREF="#scroll4">4. Fluorescence <ul id="list2"><A HREF="#scroll4.1">4.1. TOPRO3 intercalating dye</ul> <ul id="list2"><A HREF="#scroll4.2">4.2. Lysotracker Green dye</ul> <A HREF="#scroll5">5. Cells <ul id="list2"><A HREF="#scroll5.1">5.1. Cell splitting</ul> <ul id="list2"><A HREF="#scroll5.2">5.2. Cell counting</ul> <ul id="list2"><A HREF="#scroll5.3">5.3. Total DNA extraction<A NAME="scroll1"></A>.</ul>

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<div id=1> <h2><A NAME="scroll1.1"></A>1. DNA origami structure production</h2> <h3><u>1.1 Pre-Stocks</u></h1> The oligonucleotide staples required for DNA origami structure production generally come in 96 well plates, with a single oligonucleotide in each well.

</br></br>The purpose of creating pre-stocks is to segregate all staples necessary to assemble a particular section of the structure into one stock, to make folding reaction preparation more convenient in the future.

</br></br>To create pre-stocks: <ul id="list2">1. Determine which staples can be combined into one stock, and <ul id="list2">allocate wells for each stock in a 96-well plate.</ul> </ul><ul id="list2">2. Centrifuge the oligonucleotide plates at 2000 rpm for 2 minutes. </ul><ul id="list2">3. Pipet the desired oligonucleotides into the stock well. <ul id="list2">a. For 1-2 oligos, use 20 uL.</ul> <ul id="list2">b. For 2-10 oligos, use 15 uL.</ul> <ul id="list2">c. For >10 oligos, use 10 uL.</ul> <ul id="list2">d. For >30 oligos, use a trough and a multichannel pipet to mix <ul id="list2">different oligos before transferring into well.</ul></ul></ul></ul> <ul id="list2"><A NAME="scroll1.2"></A>4. Mix the contents in each pre-stock well thoroughly by pipetting.</ul>

<h3><u>1.2 Working stocks</u></h1> A working stock is the combination of all the oligonucleotide staples in the correct proportion necessary to fold a DNA origami structure.

</br></br>To create working stocks: <ul id="list2">1. Determine the volume of oligos required to fold structure properly.</ul> <ul id="list2">2. Centrifuge pre-stocks plate to bring down any liquid on the sides or<ul id="list2"> the top.</ul></ul> <ul id="list2">3. In a 1.5 mL tube, add the correct amount of pre-stock oligonucleotides <ul id="list2">previously determined.</ul></ul> <ul id="list2">4. Vortex solution for ~5 sec.</ul> <ul id="list2"><A NAME="scroll1.3"></A>5. Centrifuge for ~10-15 sec.</ul>

<h3><u>1.3 Folding reactions</u></h1> <ul id="list2">1. Prepare working stock.</ul> <ul id="list2">2. For a 20 nM scaffold concentration in 50 uL volume with 10x staple <ul id="list2">concentration:</ul> <ul id="list2">a. Add 10 uL of 100 nM scaffold.</ul> <ul id="list2">b. Add 20 uL of 500 nM staples.</ul> <ul id="list2">c. Add 10 uL of dH2O.</ul> <ul id="list2">d. Add 5 uL of 10x FOB buffer.</ul> <ul id="list2">e. Add 5 uL of MgCl2 salt in desired concentration.</ul></ul> <ul id="list2">3. Mix by pipetting.</ul> <ul id="list2"><A NAME="scroll2"></A>4. Place in thermocycler with desired temperature cycles and time ramp.</ul> </div>

<div id=2> <h2><A NAME="scroll2.1"></A>2. Imaging</h2> <h3><u>2.1. Agarose gel electrophoresis</u></h1> For 2% Agarose gels:

</br></br><u>Large gel:</u> </br>1. Measure out 2.5g Agarose in beaker. </br>2. Add 124g 0.5x TBE buffer. </br>3. Microwave for 1-2 mins to dissolve Agarose. </br>4. Replace evaporated water with dH2O until back to 124g. </br>5. Add 1 mL of 1.375 M MgCl2. </br>6. Add 5 uL ethidium bromide (EtBr) intercalating dye.* </br>7. Swirl to mix, then add into gel tray and insert comb.

</br></br><u>Small gel:</u> </br>1. Measure out 1g Agarose in beaker. </br>2. Add 49.6g 0.5x TBE buffer. </br>3. Microwave for 1-2 mins to dissolve Agarose. </br>4. Replace evaporated water with dH2O until back to 49.6g. </br>5. Add 0.4 mL of 1.375 M MgCl2. </br>6. Add 2 uL ethidium bromide (EtBr) intercalating dye.* </br>7. Swirl to mix, then add into gel tray and insert comb.

</br></br>*Add EtBr only if imaging using UV light.

</br></br><u>Gel loading:</u> </br>1. Mix 15 uL of sample with 3 uL of 6x loading dye. <ul id="list2">a. For scaffold at 100 nM, mix 1.5 uL scaffold with 13.5 uL dH2O and 3 uL <ul id="list2">dye.</ul></ul> 2. Once gel is solid, fill gel rig with TBE buffer containing MgCl2. </br>3. Remove comb. </br>4. Add 17 uL of prepared samples into the wells in the gel. <ul id="list2">a. For DNA ladder, add 6 uL.</ul> <A NAME="scroll2.1.1"></A>5. Attach electrodes, connect to power supply, and run at 70 volts. <ul id="list2">a. Small gels can be run for ~2 hours, large gels may require more than 4 <ul id="list2">hours.</ul></ul>

<h4><u>2.1.1 UV imaging:</u></h4> 1. Ensure that the gel contains EtBr. </br>2. Place gel on a UV imaging table. <ul id="list2">a. The EtBr should make DNA fluoresce under UV radiation.</ul> 3. Use a camera to image the gel. <ul id="list2"><A NAME="scroll2.1.2"></A>a. Change the brightness, contrast, or focus to ensure that bands show <ul id="list2">up clearly in image.</ul></ul>

<h4><u>2.1.2 Typhoon imaging:</u></h4> 1. Ensure that the gel DOES NOT contain EtBr. </br>2. Determine the wavelength at which fluorescent markers are excited. </br>3. Place gel in a Typhoon 9410 imager. </br>4. Select the signal type, excitation wavelength, orientation and focal height. </br>5. <A NAME="scroll2.2"></A>Image gel.

<h3><u>2.2. Sample preparation for TEM</u></h1>

<u>UFO preparation</u> </br>1. Add 5 mL of boiling deoxygenated ddH2O to 0.1g UFO powder. </br>2. Vortex vigorously for 10 mins. The solution should be light brown. </br>3. Filter solution. It should now be clear. </br>4. Aliquot 200 uL into separate tubes. </br>5. Centrifuge tube at max speed for 5 mins on a tabletop centrifuge. </br>6. Add 1 uL of 5 M NaOH to the side of the stain solution tube. Do not add it <ul id="list3">directly into the solution.</ul> 7. Vortex immediately for 2 mins. </br>8. Centrifuge at top speed for 3 mins. </br>9. Wrap tube with foil to keep away from light

</br></br><u>UFO staining</u> </br>1. Glow-discharge the carbon-coated TEM grids to make surface hydrophilic. </br>2. Apply ~3 uL of sample solution onto carbon-coated side of grid. Let it adsorb<ul id="list3"> into the grid for 4 mins.</ul> 3. After 4 mins, use filter paper to draw off liquid from the edge of the grid. Do <ul id="list3">not touch the grid surface.</ul> 4. Immerse grid sample-side first into a droplet of 10 uL 2% UFO staining <ul id="list3">solution. Remove stain solution immediately using a filter paper.</ul> 5. Immerse grid sample-side first into a droplet of 20 uL UFO. Let the grid face <ul id="list3">soak in the staining solution for 40 sec. Afterward, remove the solution using a filter paper.</ul> <A NAME="scroll2.3"></A>6. Let the grid dry for at least 15 mins before inserting into the TEM.

<h3><u>2.3. Sample preparation for TIRF</u></h1> <u>TIRF 8-well cell plate preparation</u> </br>1. Remove ~2 mL of cell suspension from stock suspension. <ul id="list2">a. equalize volume in stock with warm (37 C) human RPMI</ul> 2. Count cells in cell suspension (see protocol). </br>3. Aliquot 1x105 cells into an epi tube. </br>4. Wash cells in PBS twice. <ul id="list2">a. Centrifuge cells at 300 G for 5 mins. <ul id="list2">i. When putting tube in the centrifuge, ensure that the hinge of the <ul id="list2">cap is facing outwards, so that cell pellet will always form by the<ul id="list2">hinge.</ul></ul></ul></ul> <ul id="list2">b. Aspirate out as much as the supernatant as possible without sucking<ul id="list2"> up pellet.</ul></ul> <ul id="list2">c. Resuspend cell pellet by adding 500 uL of PBS. <ul id="list2">i. Dispense liquid by the rim of the tube.</ul></ul> <ul id="list2">d. Vortex for <1 sec to mix in pellet.</ul> <ul id="list2">e. Centrifuge at 300 G for 5 mins to form pellet again.</ul> <ul id="list2">f. Aspirate to remove supernatant.</ul> 5. While washing cells, add poly-L-lysine onto a circular plate until the surface<ul id="list3"> of the inner circle is covered.</ul> 6. Let the poly-L-lysine incubate for 5 minutes, and then wash twice with PBS<ul id="list3"> solution.</ul> <ul id="list2">a. To wash, remove the excess poly-L-lysine, add similar amount of PBS <ul id="list3"> on the plate, and remove after a few seconds.</ul></ul> 7. Stain washed cells with fluorescent dye of choice according to protocol. </br>8. Centrifuge at 300 G for 5 mins, and remove supernatant. </br>9. Wash cells twice in clear media and resuspend in same media. </br>10. Pipet ~400 uL of cell suspension onto the plate. </br>11. Add PBS to surrounding cells to prevent evaporation of sample.

</br></br><u>TIRF microscope's lide preparation</u> </br>1. Take a microscope slide, and place it at a slight incline. </br>2. Place 2 strips of double sided tape to form a channel in between the 2 pieces <ul id="list3">of tape.</ul> 3. Place a cleaned coverslip on the pieces of tape. </br>4. Press down on the tape with a small epi tube to seal the channel on the <ul id="list2">sides.</ul> 5. Pipet ~20 uL of dilute strep into the channel. <ul id="list2">a. Place the tip of the pipet by the opening between slide and coverslip and pipet slowly. The liquid should move into the channel by capillary action, helped along by the incline. Once the whole channel is full, stop pipetting.</ul> 6. Let the strep incubate for 5 minutes. </br>7. Wash excess strep away using PBS buffer. <ul id="list2">a. Pipet in PBS from the top and hold a piece of filter paper at the bottom to wick away the excess liquid being displaced on the other end. Try to match the speed of pipetting to the speed of absorption by the filter paper to prevent formation of bubbles. If a bubble blocks flow on one end, pipet from the other end to force bubble out. Small bubbles don’t really matter.</ul> 8. Pipet ~20 uL of BSA/casein into the channel. <ul id="list2">a. Same procedure as previous step.</ul> 9. Let the BSA/casein incubate for 5 minutes. </br>10. Wash with PBS buffer. </br>11. Pipet ~20 uL of structures. </br>12. Incubate for 5 minutes. </br>13. Wash with PBS buffer. </br><A NAME="scroll3"></A>14. Seal off the ends of the channel.

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<div id=3> <h2><A NAME="scroll3.1"></A>3. Purification</h2> <h3><u>3.1. Agarose gel electrophoresis</u></h1> For gel preparation, please see section 2.1. </br></br><u>Gel purification:</u> </br>1. Ensure that gel contains EtBr. </br>2. Place gel on UV imaging table. </br>3. Cut out gel pieces that display DNA fluorescence and may correspond to <ul id="list3">structures.</ul> 4. Place gel pieces into the filter of a freeze n’ squeeze tube. </br>5. Centrifuge at 13,000 g for 6 mins. </br><A NAME="scroll3.2"></A>6. Remove filter and store liquid containing purified structures.

<h3><u>3.2. PEG purification</u></h1> 1. In a 1.5 mL tube, add the desired volume of structure. </br>2. Add the same volume of 15% PEG 8000. Vortex well to mix. </br>3. Centrifuge at 16,000 g for 30 mins. </br><A NAME="scroll4"></A>4. Resuspend in buffer of choice.

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<div id=4> <h2><A NAME="scroll4.1"></A>4. Fluorescence</h2> <h3><u>4.1. TOPRO3 intercalating dye</u></h1> 1. Calculate the concentration of structure in desired volume. </br>2. Add correct volume of intercalating dye into structure solution. </br>3. For 1 TOPRO3 molecule every 10 base pairs from a stock of 1 mM dye: </br></br> (volume of origami (μL) × concentration origami (nM) × length of scaffold(bp) ×〖 10〗^(-7))

</br>= TOPRO3 (μL) </br></br>1. Combine desired amount of origami with calculated TOPRO3. </br>2. Let sit for 2-24 hours: can speed up process by placing in shaker/incubator at <ul id="list3">37oC and shake. </ul> 3. Centrifuge at 16,000 xg+ for 25 minutes. Should have visible pellet. </br>4. Remove supernatant/ discard </br><A NAME="scroll4.2"></A>5. Resuspend pellet with desired buffer (same volume as before)

<h3><u>4.2. Lysotracker Green dye</u></h1>

1. Count Cells </br>2. Get volume for 500,000 cells. </br>3. Wash twice with PBS. </br>4. Add lysotracker dye to media: <ul id="list2">a. Concentration of bottle of lysotracker = 1 mM</ul> <ul id="list2">b. Final concentration in media = 100 nM</ul> 5. Warm media+dye to 37 degrees </br>6. Add warm media to cells </br>7. Let cells incubate in dye+media for 1.5 hours </br><A NAME="scroll5"></A>8. Centrifuge and resuspend cells in clear media before imaging

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<div id=5> <h2><A NAME="scroll5.1"></A>5. Cells</h2> <h3><u>5.1. Cell splitting</u></h1> 1. Pipet ~10 mL of Human RPMI from the stock solution into a falcon tube <ul id="list3">using a serological pipet and let it warm up to 37 C in the incubator.</ul> 2. Take the cells out of the refrigerator and observe them under the <ul id="list3">microscope to see how the cells are doing.</ul> 3. Remove 8 mL of the cell media from the flask. <ul id="list2">a. Make sure to mix vigorously before removing the media.</ul> <ul id="list2">b. The ideal way to do this is to suck up all the media in the flask, then deposit 2 mL back into the flask. This way, we are always sure of the amount of old media in the flask.</ul> 4. Pipes 8 mL of the warm human RPMI into the flask, and pipet a few times to <ul id="list3">ensure proper mixing.</ul> <A NAME="scroll5.2"></A>5. Store the flask back into the incubator.

<h3><u>5.2. Cell counting</u></h1>

1. Transfer 10 mL of cells into a 15 mL falcon tube. </br>2. Add 10 uL of dye (Trypan - Blue) to an eppendorf tube </br>3. Add 10 uL of cells to the same tube. <ul id="list2">a. Should be a 1:1 ratio of cells and dye</ul> 4. Add 10 uL of the mixture to the hemocytometer under the cover slip. </br>5. Put the hemocytometer on the scope, turn the scope on, focus image. </br>6. Count the cells in the 4 corners of the cover slip. </br>7. Calculate the number of cells: (total from 4 corners)/4*2*10,000 = total <ul id="list3">cells/mL</ul> <A NAME="scroll5.3"></A>8. Wipe coverslip and hemocytometer with ethanol.

<h3><u>5.3. Total DNA extraction</u></h1>

1. Spin down the cells and remove supernatant. </br>2. Wash cells once with PBS. </br>3. Resuspend cells by adding 200 uL PBS buffer. </br>4. Add 20 uL of proteinase K (in kit). </br>5. Add 200 uL of buffer AL (in kit). Mix thoroughly by pulse vortexing for 5-10 <ul id="list3">seconds. Incubate at 56 C for 10 mins.</ul> 6. Add 200 uL (96-100%) ethanol to solution. Mix thoroughly by pulse vortexing. </br>7. Pipet the mixture into DNeasy mini spin column (in kit) and place in a 2 mL <ul id="list3">collection tube (in kit). Centrifuge at 6000 g for 1 min.</ul> 8. Discard the liquid in the collection tube. </br>9. Place the spin column into a new collection tube, add 500 uL of Buffer AW1, <ul id="list3">and centrifuge at 6000 g for 1 min. </ul> 10. Discard the liquid in the collection tube. </br>11. Place the spin column into a new collection tube, add 500 uL of Buffer AW2, <ul id="list3">and centrifuge at 6000 g for 1 min. Discard the liquid in the collection tube.</ul> 12. Place the spin column into a 1.5 mL epi tube, and pipet 200 uL Buffer AE<ul id="list3"> directly onto the membrane. </ul> 13.Let it incubate at room temperature for 1 min, then centrifuge at 6000 g for <ul id="list3">1 minute.</ul> 14. The liquid in the epi tube will contain total DNA from cells.

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Biomod/2014/OhioMOD/protocols: Difference between revisions

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@@ Line 69: / Line 69: @@
 <ul id="list2"><A HREF="#scroll5.1">5.1.	Cell splitting</ul>
 <ul id="list2"><A HREF="#scroll5.2">5.2.	Cell counting</ul>
-<ul id="list2"><A HREF="#scroll5.3">5.3.	Total DNA <A NAME="scroll1"></A>extraction</ul>
+<ul id="list2"><A HREF="#scroll5.3">5.3.	Total DNA extraction<A NAME="scroll1"></A>.</ul>
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