iGEM 2014 — Team UC-Santa_Cruz-BioE

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# Team:UC-Santa Cruz-BioE/Notebook

### From 2014.igem.org

Notebook

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# Important Protocols

### Glycerol Stock Preparation

* 1. Inoculated TSB with MR-1 (either from plate or glycerol stock) and grow overnight.
* 2. add glycerol to culture to final concentration of 80% glycerol.
* 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).

### Microbial Fuel Cell Assembly

* 1. cut two 3 inch PVC tubes .
* 2. sandwich them with nafion cation exchange membrane between them.
* 3. add caulking around both sides of membrane-tube junction.
* 4. let dry for 15 minutes.
* 5. repeat step 3 and 4.
* 6. cut off excess membrane around junction.
* 7. add one more layer to seal up junction.
* 8. let dry overnight.
* 9. cut 2.5in-3in x 12in of carbon cloth
* 10. roll around tip of finger.
* 11. weave titanium wire close to end edge of roll.
* 12 fold titanium wire up the length of the roll.
* 13. twist excess wire around itself.
* 14. cut wire at .5in-1in.
* 15. push through stopper.
* 16. seal hole from wire with caulking.
* 17. let dry overnight. (two of these make up the cathode and the anode)
* 18. pour potassium ferricyanide in one chamber of the cell.
* 19. carefully push stopper with carbon cloth to plug the chamber.
* 20. push hollow needle through stopper to let air out of chamber.
* 21. pour overnight culture of MR-1 into other chamber.
* 22. place glass slide in chamber.
* 23. carefully push stopper with carbon cloth to plug the chamber.
* 24. push new sterile hollow needle in stopper into air bubble in chamber to let out any CO2 bacteria will produce.
* 25. open ExcelINX up on computer.
* 26. connect electrodes from DMM to cathode and anode (remember which channels are being used).
* 27. start ExcelINX.

### Plasmid Prep

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.

* 1. Grow culture of E.coli in TSB (or LB) overnight.
* 2. transfer culture to eppendorf microcentrifuge tubes.
* 3. spin at 10,000-14,000 rpm for 10 mins at room temperature.
* 4. remove supernatant and add 200ml of resuspension buffer to pellet.
* 5. add 5ul of RNase to suspension.
* 6. incubate suspension at room temperature for 5 minutes.
* 7. add 350ul of Lysis Buffer and mix by inversion 4-6 times (Do not vortex to avoid breaking plasmid).
* 8. add 200ul of potassium acetate and mix thoroughly by inversion. White precipitate should form.
* 9. Centrifuge tube at full speed for 10 minutes.
* 10. carefully remove supernatant and put in new tube. Be sure to avoid pipetting the white precipitate.
* 11. add 0.6 volume of 100% ethanol to supernatant.
* 12. mix by inversion 4-6 times and let sit at room temperature for 10 minutes.
* 13. centrifuge at full speed for 5 minutes.
* 14. remove supernatant and add 70% ethanol.
* 15. centrifuge at full speed for 3 minutes.
* 16. let tubes sit for 10 minutes to let some ethanol evaporate.
* 17. discard supernatant.
* 18. add 60ul of TE buffer and use nanodrop to check purity and concentration of sample.
* 19. store in -20Cor -80C freezer.

### Conjugation Protocol

We used this protocol for  [conjugation](https://static.igem.org/mediawiki/2012/0/0f/Shewanella_Conjugation.pdf)(credits to Dylan Webster and Dr.Jeff Gralnick)

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