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Team:UC-Santa Cruz-BioE/Notebook
From 2014.igem.org
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<li>1. Grow culture of E.coli in TSB (or LB) overnight.</li> | <li>1. Grow culture of E.coli in TSB (or LB) overnight.</li> | ||
Latest revision as of 03:30, 18 October 2014
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(credits to Dylan Webster and Dr.Jeff Gralnick)
