Biomod/2014/ASU/Protocols

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1 Biomod/2014/ASU

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Biomod/2014/ASU
		
			From OpenWetWare
			< <a href="/wiki/Biomod" title="Biomod">Biomod</a> | <a href="/wiki/Biomod/2014" title="Biomod/2014">2014</a>
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<nav id = "main-nav">

		
			<a href="http://openwetware.org/wiki/Biomod/2014/ASU">Main</a>
			<a href="http://openwetware.org/wiki/Biomod/2014/ASU/Project">Project</a>
			<a href="http://openwetware.org/wiki/Biomod/2014/ASU/Design">Design</a>
			<a href="#">Experiment</a>
                                 
                                       <a href = "http://openwetware.org/wiki/Biomod/2014/ASU/Materials">Materials</a>
                                       <a href = "http://openwetware.org/wiki/Biomod/2014/ASU/Protocols">Protocols</a>
                                 
                        <a href="http://openwetware.org/wiki/Biomod/2014/ASU/Results">Results</a>
                        <a href="http://openwetware.org/wiki/Biomod/2014/ASU/Discussion">Discussion</a>
			<a href="http://openwetware.org/wiki/Biomod/2014/ASU/Team">Team</a>	
                        <a href="http://openwetware.org/wiki/Biomod/2014/ASU/Acknowledgement">Acknowledgements</a>	
		
	</nav>

 
     
                
                       <img src = "http://openwetware.org/images/1/1b/Rsz_ascube.png">

                
      

Tile Assembly
                       
1. Put all relevant tile strands into a 1x TAE/Mg2+ buffered solution at 1 μM concentration.
2. Anneal tiles by slowly cooling from 90 °C to 25 °C over the course of 10 hours according to the following protocol:


<img src = "http://openwetware.org/images/2/2f/Screen_Shot_2014-10-23_at_3.23.40_AM.png">


4% Agarose gel electrophoresis
                       
1. Prepare the agarose gel chamber by inserting gel walls and comb.
2. Add 0.2 g of agarose powder to 50 mL 0.5X TBE in an Erlenmeyer flask. Gently mix by swirling.
3. Microwave the agarose solution for 45 seconds on HIGH.
4. Add 5 μL SYBR Safe stain to the heated agarose solution.
5. Pour the heated gel solution into the chamber. Allow half an hour for the gel to polymerize and harden.
6. After the gel has polymerized, remove the temporary gel walls and fill the chamber with 0.5X TBE.
7. Prepare loading samples by adding one-tenth their volume of 10X native loading dye to each.
8. Pipet 8 μL of each sample into the sample wells.
9. Run at 100V for an hour and a half or until the loading dye has migrated approximately halfway through the gel.
10. Image the gel via UV transillumination.



EMSA via Native PAGE
                       
1. Prepare loading samples
     a. Prepare 10 μL samples of 20 nM DNA tile + 1 μM alpha-thrombin in a 1x TAE/Mg2+ +100 mM KCl buffered solution.
     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 
     experimental sample above.
     c. Allow to incubate on the benchtop at 25 °C for 1 hour. 
     d. Add 1.1 μL 10x native dye to each sample and mix gently with a pipette.
2. While samples are incubating, prepare an 8% Native PAGE gel:
     a. Set up the gel assembly by aligning glass plates and spacers, tightening the screws of the clamps, screwing the assembly down
     into a rubber pad, and inserting a comb.
     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.
     c. Add 16.8 μL TEMED and 300 μL APS. Swirl to mix. 
     d. Use a serological pipette to fill the assembly with gel solution. Allow 40 minutes for polymerization.
     e. Remove the comb and attach an upper buffer chamber to the gel assembly and to a second, solid glass plate filler.
     f. Fill buffer chamber with 500 mL 1x TAE/Mg2+ + 100 mM KCl. Rinse out wells of gel with ambient buffer using a needled
      syringe.
3. Load and run the gel:
     a. Load 10 μL sample into different wells of the gel.
     b. Put the completed gel assembly into a lower buffer chamber with 1x TAE/Mg2+. Connect it to a voltage generator, and run the
      gel at 200 V for 8 hours.
     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.
4. Stain the gel:
     a. Mix 10 uL 10000x SYBR Green with 100 mL water in a clean staining box.
     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 
     evenly stained. Allow the gel to stain for 5 minutes.
     c. After staining, briefly place the gel in water to destain.
5. Image the gel:
     a. Place the gel on the transilluminating plate of the gel imager.
     b. Image the gel using UV transillumination.


PCR
                       
1. Prepare a PCR master mix solution with 2 extra equivalents. For 20 μL reactions, add, for each equivalent:
     a. 4 μL 5x Phusion buffer.
     b. 0.4 μL 10 mM dNTPs.
     c. 0.6 μL DMSO.
     d. Forward and reverse primers to 1 μM.
     e. 0.2 μL Phusion High-Fidelity DNA polymerase (keep on ice)
          i. Add polymerase last and keep all mixtures containing polymerase on ice thereafter.
2. Add 2 μL of each target oligonucleotide source to 18 μL of master mix for each reaction. Keep on ice.
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:
     1. 98 °C for 1 minute
     2. 98 °C for 5 seconds
     3. 42 °C for 10 seconds
     4. 72 °C for 15 seconds
     4. Cycle steps 2-4 for 24 more times
     5. 72 °C for 5 minutes
     6. 4 °C forever





Alpha-Thrombin-bound NHS ester bead affinity column:
                       
1. Prepare columns by functionalizing Pierce® NHS-Activated Agarose Dry Resin with alpha-thrombin:
     a. In one pot, measure out 5 mg Pierce® NHS-Activated Agarose Dry Resin per each column to 
      be prepared.
     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 be prepared.
     c. Combine entire diluted alpha-thrombin stock with weighed-out Pierce® NHS-Activated Agarose Dry Resin.
     d. Mix end-over-end for one hour in a filter column.
     e. Centrifuge at 1000 rfc for 1 minute. Save flowthrough.
     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 
     flowthrough.
     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.
     h. Centrifuge at 1000 rfc for 1 minute and save flowthrough.
     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 
     quenching.
     j. Split mix into columns with 30 μL suspended beads each and store at 4 °C.



2. Run column assay:
     a. Prepare a 1 μM loading solution of the desired sample in 20 μL of a 1x TAE + 0.1M KCl buffered solution.
     b. Add 60 μL 1x TAE + 0.1M KCl to column, and mix by gently pipetting. Centrifuge at 1000 rfc for 1 minute.
     c. Add 20 μL loading solution to column. Mix end-over-end for 1 hour.
     d. Centrifuge at 1000 rfc for 1 minute, and save flowthrough in an Eppendorf tube.
     e. Add 60 μL 1x TAE + 0.1M KCl to column, and mix by gently pipetting.
     f. Centrifuge at 1000 rfc for 1 minute, and save flowthrough in an Eppendorf tube.
     g. Repeat steps e-f 3 more times. 
     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 
     storage.
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.







			
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Biomod/2014/ASU/Protocols

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