<p>All tubes will have Milli-Q water and reaction buffer. The labels above indicate what will be added to each tube for the reaction.</p>
</li>
<li>Add Milli-Q water, Cas13 reaction buffer, crRNA, and Cas13 in the order listed to the appropriate Eppendorf tube.</li>
<li>Incubate the solution at 37 C for 1 hour in a water bath.</li>
<li>Remove the tube from the water bath and add 9 uL of miRNA to the solution as well as enough Milli-Q to bring it to 20 uL and mix carefully by gently inverting the tube.</li>
<li>Incubate the solution again at 37 C for 10 minutes.</li>
</ol>
<br>
<h2>Protocol for Current Gel Electrophoresis Method:</h2>
<ol>
<li>Measure 2 g agarose powder and add it to a 250 mL flask.</li>
<li>Add 50 mL 1x TAE buffer to the flask.</li>
<li>Dissolve the agarose in a microwave or hot (>95°C) water bath until the solution becomes clear. If using a microwave, heat the solution for several short intervals. The agarose is completely dissolved when the solution is completely clear.</li>
<li>Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly. The solution is cool enough when you can grab the flask with your bare hand.</li>
<li>Add 2 μL of ethidium bromide to the solution and swirl to mix. Wear gloves when handling ethidium bromide and whenever handling the gel for the rest of the protocol.</li>
<li>Secure the rails on either side of the gel casting tray in the up position such that the gel will not leak out of the tray as it cools.</li>
<li>Pour the melted agarose solution into the casting tray. Place the comb in the comb slot near the end of the casting tray.</li>
<li>Let the gel cool until it is solid (10-20 minutes). It will become opaque as it cools.</li>
<li>Carefully unscrew and lower the rails on the gel box.</li>
<li>Place the gel in the electrophoresis chamber with the wells on the negative (black) end of the gel box. Add enough TAE buffer so that the gel is completely submerged by at least 2-3 mm.</li>
<li>Carefully and slowly pull the comb straight up and out of the gel.</li>
<li><p>Prepare a chart to keep track of which sample you will place in each well.</p><table><tr><th>RNA Ladder</th><th>purified crRNA</th><th>Purified miRNA</th><th>CAS13</th><th>crRNA + CAS13</th><th>miRNA + CAS13</th><th>miNA + crRNA</th><th>miRNA + crRNA + CAS13</th></tr></table></li>
<li>Add 2 μL loading dye per 10 μL sample and mix thoroughly by pipetting up and down.</li>
<li>Before adding to the gel, the samples must be denatured to prevent the formation of secondary structures.<ol><li>Incubate in a 70°C water bath for 10 minutes.</li><li>Chill samples on ice for 3 minutes and spin briefly.</li></ol></li>
<li>Add 10 μL of each sample to a well using one sample per well, exhibiting caution.</li>
<li>Run the gel immediately so that the RNA samples do not diffuse out of the wells.</li>
<li>Put the gel on a UV illuminator to see the results.</li>
