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<span style="color:#191970;font-family:Arial;font-size:16px;"><h1>Important Protocols</h1><!..A HEADING!..></span></div>
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<h3>Glycerol Stock Preparation</h3>
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<ul>
<li>1. Inoculated TSB with MR-1 (either from plate or glycerol stock) and grow overnight.</li>
<li>2. add glycerol to culture to final concentration of 80% glycerol.</li>
<li>3. place 1ml in 1.5ml freezer safe eppendorf tubes (do not fill eppendorfs with more than 1.5ml to allow for expansion by freezing).</li>
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<h3>Microbial Fuel Cell Assembly</h3>
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<li>1. cut two 3 inch PVC tubes .</li>
<li>2. sandwich them with nafion cation exchange membrane between them.</li>
<li>3. add caulking around both sides of membrane-tube junction.</li>
<li>4. let dry for 15 minutes.</li>
<li>5. repeat step 3 and 4.</li>
<li>6. cut off excess membrane around junction.</li>
<li>7. add one more layer to seal up junction.</li>
<li>8. let dry overnight.</li>
<li>9. cut 2.5in-3in x 12in of carbon cloth</li>
<li>10. roll around tip of finger.</li>
<li>11. weave titanium wire close to end edge of roll.</li>
<li>12 fold titanium wire up the length of the roll.</li>
<li>13. twist excess wire around itself.</li>
<li>14. cut wire at .5in-1in.</li>
<li>15. push through stopper.</li>
<li>16. seal hole from wire with caulking.</li>
<li>17. let dry overnight. (two of these make up the cathode and the anode)</li>
<li>18. pour potassium ferricyanide in one chamber of the cell.</li>
<li>19. carefully push stopper with carbon cloth to plug the chamber.</li>
<li>20. push hollow needle through stopper to let air out of chamber.</li>
<li>21. pour overnight culture of MR-1 into other chamber.</li>
<li>22. place glass slide in chamber.</li>
<li>23. carefully push stopper with carbon cloth to plug the chamber.</li>
<li>24. push new sterile hollow needle in stopper into air bubble in chamber to let out any CO2 bacteria will produce.</li>
<li>25. open ExcelINX up on computer.</li>
<li>26. connect electrodes from DMM to cathode and anode (remember which channels are being used).</li>
<li>27. start ExcelINX.</li>
</ul>
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<h3>Plasmid Prep</h3>
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<p>This is the protocol for DNA extraction and Purification using the Edvotek 202 Plasmid Prep Kit. All reagents except Isopropanol and Ethanol were provided with the kit.
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<ul>
<li>1. Grow culture of E.coli in TSB (or LB) overnight.</li>
<li>2. transfer culture to eppendorf microcentrifuge tubes.</li>
<li>3. spin at 10,000-14,000 rpm for 10 mins at room temperature.</li>
<li>4. remove supernatant and add 200ml of resuspension buffer to pellet.</li>
<li>5. add 5ul of RNase to suspension.</li>
<li>6. incubate suspension at room temperature for 5 minutes.</li>
<li>7. add 350ul of Lysis Buffer and mix by inversion 4-6 times (Do not vortex to avoid breaking plasmid).</li>
<li>8. add 200ul of potassium acetate and mix thoroughly by inversion. White precipitate should form. </li>
<li>9. Centrifuge tube at full speed for 10 minutes.</li>
<li>10. carefully remove supernatant and put in new tube. Be sure to avoid pipetting the white precipitate. </li>
<li>11. add 0.6 volume of 100% ethanol to supernatant.</li>
<li>12. mix by inversion 4-6 times and let sit at room temperature for 10 minutes.</li>
<li>13. centrifuge at full speed for 5 minutes.</li>
<li>14. remove supernatant and add 70% ethanol.</li>
<li>15. centrifuge at full speed for 3 minutes.</li>
<li>16. let tubes sit for 10 minutes to let some ethanol evaporate.</li>
<li>17. discard supernatant.</li>
<li>18. add 60ul of TE buffer and use nanodrop to check purity and concentration of sample.</li>
<li>19. store in -20Cor -80C freezer.</li>
</ul>
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<h3>Conjugation Protocol</h3>
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<p>We used this protocol for <a href="https://static.igem.org/mediawiki/2012/0/0f/Shewanella_Conjugation.pdf"> conjugation</a>(credits to Dylan Webster and Dr.Jeff Gralnick)</p>
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