Biomod/2014/ASU/Protocols
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<p class = "serif" style = "color: #000000; font-size: 25px;"><span class = "mw-headline">Tile Assembly</span></p>
<hr>
<p style = "color: #000000; font-size: 16px;">1. Put all relevant tile strands into a 1x TAE/Mg2+ buffered solution at 1 μM concentration.</p>
<p style = "color: #000000; font-size: 16px;">2. Anneal tiles by slowly cooling from 90 °C to 25 °C over the course of 10 hours according to the following protocol:</p><br>
<img src = "http://openwetware.org/images/2/2f/Screen_Shot_2014-10-23_at_3.23.40_AM.png"><br>
<p class = "serif" style = "color: #000000; font-size: 25px;"><span class = "mw-headline">4% Agarose gel electrophoresis</span></p>
<hr>
<p style = "color: #000000; font-size: 16px;">1. Prepare the agarose gel chamber by inserting gel walls and comb.</p>
<p style = "color: #000000; font-size: 16px;">2. Add 0.2 g of agarose powder to 50 mL 0.5X TBE in an Erlenmeyer flask. Gently mix by swirling.</p>
<p style = "color: #000000; font-size: 16px;">3. Microwave the agarose solution for 45 seconds on HIGH.</p>
<p style = "color: #000000; font-size: 16px;">4. Add 5 μL SYBR Safe stain to the heated agarose solution.</p>
<p style = "color: #000000; font-size: 16px;">5. Pour the heated gel solution into the chamber. Allow half an hour for the gel to polymerize and harden.</p>
<p style = "color: #000000; font-size: 16px;">6. After the gel has polymerized, remove the temporary gel walls and fill the chamber with 0.5X TBE.</p>
<p style = "color: #000000; font-size: 16px;">7. Prepare loading samples by adding one-tenth their volume of 10X native loading dye to each.</p>
<p style = "color: #000000; font-size: 16px;">8. Pipet 8 μL of each sample into the sample wells.</p>
<p style = "color: #000000; font-size: 16px;">9. Run at 100V for an hour and a half or until the loading dye has migrated approximately halfway through the gel.</p>
<p style = "color: #000000; font-size: 16px;">10. Image the gel via UV transillumination.</p>
<br>
<p class = "serif" style = "color: #000000; font-size: 25px;"><span class = "mw-headline">EMSA via Native PAGE</span></p>
<hr>
<p style = "color: #000000; font-size: 16px;text-decoration:underline;">1. Prepare loading samples</p>
<p style = "color: #000000; font-size: 16px;"> a. Prepare 10 μL samples of 20 nM DNA tile + 1 μM alpha-thrombin in a 1x TAE/Mg2+ +100 mM KCl buffered solution.</p>
<p style = "color: #000000; font-size: 16px;"> b. Prepare a negative control 10 μL sample of 20 nM DNA tile in a 1x TAE/Mg2+ + 100 mM KCl buffered solution for each</p>
<p style = "color: #000000; font-size: 16px;"> experimental sample above.</p>
<p style = "color: #000000; font-size: 16px;"> c. Allow to incubate on the benchtop at 25 °C for 1 hour. </p>
<p style = "color: #000000; font-size: 16px;"> d. Add 1.1 μL 10x native dye to each sample and mix gently with a pipette.</p>
<p style = "color: #000000; font-size: 16px;text-decoration:underline;">2. While samples are incubating, prepare an 8% Native PAGE gel:</p>
<p style = "color: #000000; font-size: 16px;"> a. Set up the gel assembly by aligning glass plates and spacers, tightening the screws of the clamps, screwing the assembly down</p>
<p style = "color: #000000; font-size: 16px;"> into a rubber pad, and inserting a comb.</p>
<p style = "color: #000000; font-size: 16px;"> b. Mix 28 mL H2O, 4 mL 10x TAE/Mg2+ + 1M KCl buffer, and 8 mL 40% acrylamide in an Erlenmeyer flask. Swirl to mix.</p>
<p style = "color: #000000; font-size: 16px;"> c. Add 16.8 μL TEMED and 300 μL APS. Swirl to mix. </p>
<p style = "color: #000000; font-size: 16px;"> d. Use a serological pipette to fill the assembly with gel solution. Allow 40 minutes for polymerization.</p>
<p style = "color: #000000; font-size: 16px;"> e. Remove the comb and attach an upper buffer chamber to the gel assembly and to a second, solid glass plate filler.</p>
<p style = "color: #000000; font-size: 16px;"> f. Fill buffer chamber with 500 mL 1x TAE/Mg2+ + 100 mM KCl. Rinse out wells of gel with ambient buffer using a needled</p>
<p style = "color: #000000; font-size: 16px;"> syringe.</p>
<p style = "color: #000000; font-size: 16px;text-decoration:underline;">3. Load and run the gel:</p>
<p style = "color: #000000; font-size: 16px;"> a. Load 10 μL sample into different wells of the gel.</p>
<p style = "color: #000000; font-size: 16px;"> b. Put the completed gel assembly into a lower buffer chamber with 1x TAE/Mg2+. Connect it to a voltage generator, and run the</p>
<p style = "color: #000000; font-size: 16px;"> gel at 200 V for 8 hours.</p>
<p style = "color: #000000; font-size: 16px;"> c. After the gel has run for four hours, refresh the upper buffer chamber with 500 mL of fresh 1x TAE/Mg2+ + 100 mM KCl buffer.</p>
<p style = "color: #000000; font-size: 16px;text-decoration:underline;">4. Stain the gel:</p>
<p style = "color: #000000; font-size: 16px;"> a. Mix 10 uL 10000x SYBR Green with 100 mL water in a clean staining box.</p>
<p style = "color: #000000; font-size: 16px;"> b. Disassemble the gel apparatus, and place the gel in the stain. Briefly and gently rock the staining box to ensure the gel is being </p>
<p style = "color: #000000; font-size: 16px;"> evenly stained. Allow the gel to stain for 5 minutes.</p>
<p style = "color: #000000; font-size: 16px;"> c. After staining, briefly place the gel in water to destain.</p>
<p style = "color: #000000; font-size: 16px;text-decoration:underline;">5. Image the gel:</p>
<p style = "color: #000000; font-size: 16px;"> a. Place the gel on the transilluminating plate of the gel imager.</p>
<p style = "color: #000000; font-size: 16px;"> b. Image the gel using UV transillumination.</p><br>
<p class = "serif" style = "color: #000000; font-size: 25px;"><span class = "mw-headline">PCR</span></p>
<hr>
<p style = "color: #000000; font-size: 16px;text-decoration:underline;">1. Prepare a PCR master mix solution with 2 extra equivalents. For 20 μL reactions, add, for each equivalent:</p>
<p style = "color: #000000; font-size: 16px;"> a. 4 μL 5x Phusion buffer.</p>
<p style = "color: #000000; font-size: 16px;"> b. 0.4 μL 10 mM dNTPs.</p>
<p style = "color: #000000; font-size: 16px;"> c. 0.6 μL DMSO.</p>
<p style = "color: #000000; font-size: 16px;"> d. Forward and reverse primers to 1 μM.</p>
<p style = "color: #000000; font-size: 16px;"> e. 0.2 μL Phusion High-Fidelity DNA polymerase (keep on ice)</p>
<p style = "color: #000000; font-size: 16px;"> i. Add polymerase last and keep all mixtures containing polymerase on ice thereafter.</p>
<p style = "color: #000000; font-size: 16px;text-decoration:underline;">2. Add 2 μL of each target oligonucleotide source to 18 μL of master mix for each reaction. Keep on ice.</p>
<p style = "color: #000000; font-size: 16px;text-decoration:underline;">3. Except for a PCR-negative control, which is kept at 4 °C for the duration of thermocycling, cycle all reactions according to the following protocol:</p>
<p style = "color: #000000; font-size: 16px;"> 1. 98 °C for 1 minute</p>
<p style = "color: #000000; font-size: 16px;"> 2. 98 °C for 5 seconds</p>
<p style = "color: #000000; font-size: 16px;"> 3. 42 °C for 10 seconds</p>
<p style = "color: #000000; font-size: 16px;"> 4. 72 °C for 15 seconds</p>
<p style = "color: #000000; font-size: 16px;"> 4. Cycle steps 2-4 for 24 more times</p>
<p style = "color: #000000; font-size: 16px;"> 5. 72 °C for 5 minutes</p>
<p style = "color: #000000; font-size: 16px;"> 6. 4 °C forever</p><br>
<p class = "serif" style = "color: #000000; font-size: 25px;"><span class = "mw-headline">Alpha-Thrombin-bound NHS ester bead affinity column:</span></p>
<hr>
<p style = "color: #000000; font-size: 16px;">1. Prepare columns by functionalizing Pierce® NHS-Activated Agarose Dry Resin with alpha-thrombin:</p>
<p style = "color: #000000; font-size: 16px;"> a. In one pot, measure out 5 mg Pierce® NHS-Activated Agarose Dry Resin per each column to be prepared.</p>
<p style = "color: #000000; font-size: 16px;"> b. Dilute alpha-thrombin stock into a 1 mg/mL 0.1 M HEPES + 0.15 M NaCl buffered solution with 30 μL volume per each column to</p>
<p style = "color: #000000; font-size: 16px;"> be prepared.</p>
<p style = "color: #000000; font-size: 16px;"> c. Combine entire diluted alpha-thrombin stock with weighed-out Pierce® NHS-Activated Agarose Dry Resin.</p>
<p style = "color: #000000; font-size: 16px;"> d. Mix end-over-end for one hour in a filter column.</p>
<p style = "color: #000000; font-size: 16px;"> e. Centrifuge at 1000 rfc for 1 minute. Save flowthrough.</p>
<p style = "color: #000000; font-size: 16px;"> f. Add 30 μL volume per column to be prepared of 0.1 M HEPES, 0.15 M NaCl buffer. Recentrifuge at 1000 rfc for 1 minute and save</p>
<p style = "color: #000000; font-size: 16px;"> flowthrough.</p>
<p style = "color: #000000; font-size: 16px;"> g. Add 30 μL per column to be prepared of Tris buffer at pH 8 to quench column and mix end-over-end for 15-20 minutes at room temperature.</p>
<p style = "color: #000000; font-size: 16px;"> h. Centrifuge at 1000 rfc for 1 minute and save flowthrough.</p>
<p style = "color: #000000; font-size: 16px;"> i. Add 30 μL volume per column to be prepared of 0.1 M HEPES + 0.15 M NaCl + 0.05% NaN3 buffer to resuspend resin after</p>
<p style = "color: #000000; font-size: 16px;"> quenching.</p>
<p style = "color: #000000; font-size: 16px;"> j. Split mix into columns with 30 μL suspended beads each and store at 4 °C.</p>
<p style = "color: #000000; font-size: 16px;">2. Run column assay:</p>
<p style = "color: #000000; font-size: 16px;"> a. Prepare a 1 μM loading solution of the desired sample in 20 μL of a 1x TAE + 0.1M KCl buffered solution.</p>
<p style = "color: #000000; font-size: 16px;"> b. Add 60 μL 1x TAE + 0.1M KCl to column, and mix by gently pipetting. Centrifuge at 1000 rfc for 1 minute.</p>
<p style = "color: #000000; font-size: 16px;"> c. Add 20 μL loading solution to column. Mix end-over-end for 1 hour.</p>
<p style = "color: #000000; font-size: 16px;"> d. Centrifuge at 1000 rfc for 1 minute, and save flowthrough in an Eppendorf tube.</p>
<p style = "color: #000000; font-size: 16px;"> e. Add 60 μL 1x TAE + 0.1M KCl to column, and mix by gently pipetting.</p>
<p style = "color: #000000; font-size: 16px;"> f. Centrifuge at 1000 rfc for 1 minute, and save flowthrough in an Eppendorf tube.</p>
<p style = "color: #000000; font-size: 16px;"> g. Repeat steps e-f 3 more times. </p>
<p style = "color: #000000; font-size: 16px;"> h. Add 60 μL 1x TAE + 0.1M KCl to column, mix by gently pipetting, and pipet the contents of the column into an Eppendorf tube for</p>
<p style = "color: #000000; font-size: 16px;"> storage.</p>
<p style = "color: #000000; font-size: 16px;">Atomic Force Microscopy was performed by Shuoxing Jiang, a member of the Hao Yan Lab, using the Dimension FastScan AFM (Bruker). 2μl of sample was deposited onto freshly cleaved mica and left to absorb for 2 minutes. 100μl of 1x TAE Mg was pipetted on top of the DNA sample and the sample was imaged in ScanAsyst in Fluid mode using a ScanAssyst Fluid+ tip.</p>
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