Biomod/2014/ASU/Protocols
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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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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">
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 NHS ester beads with Alpha-Thrombin:
a. In one pot, measure out 5 mg NHS ester bead dry resin per each column to be prepared.
b. Dilute Alpha-Thrombin stock with water into a 1 mg/mL solution of volume 30 μL per each column to be prepared.
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