MOBIO experienced User Protocol

Extraction of the water filters that came from the surface waters of the Eastern Twin Mound (Above picture) went well. PCR of the DNA will take place tomorrow. The protocol for yesterday's extraction changed. I used the soil extraction kit instead of the phenol:cholorform protocol from Dr. Vesbach's lab.

Protocol:
DNA Extraction from water sample (using Mo Bio Power Soil Kit catalog #12888)

Sample should have sucrose lysis buffer added in the field and be stored at -80 degrees Celsius. Be sure to do a negative control to ensue sterile technique. Everything added to the sample should also be added to the negative control with exception of the actual sample. All centrifuge spin times are done at 10,000rcf at room temperature.

1. Place sample on ice and allow to thaw completely.
2. Add 60 uL of solution C1 to the Mo Bio Powerbead tubes. Add sample to the tube. Be sure to add as much as possible. Leave room in the top of the tube.
(C1 contains SDS and other disruption agents required for complete cell lysis. In addition to aiding in cell lysis, SDS is an anionic detergent that breaks down fatty acids and lipids associated with the cell membrane of several organisms. It if gets cold, it will form a white precipitate in the bottle. Heating to 60 degrees Celsius will dissolve the SDS and will not harm the SDS or the other disruption agents.)
3. Vortex for 1 minute.
4. Centrifuge for 30 seconds.
5. Pour supternatant to a sterile 2ml microcentrifuge tube.
6. Add 250 ul of solution C2, vortex and incubate at 4 degrees Celsius for 5 minutes.
(Solution C2 contains a reagent to precipitate non-DNA organic and inorganic material including humic substances, cell debris, and proteins; these material can contaminate the DNA purity and inhibit further analysis of the gene in question)
7. Centrifuge for 1 minute.
8. Without touching the pellet transfer 600 ul (or less) of solution to a new sterile 2 ml microtube.
9. Add 200 ul of solution C3, vortex and incubate at 4 degrees Celsius for 5 minutes.
(Solution C3 is a second reagent that removes contaminating organic and inorganic material)
10. Centrifuge for 1 minute.
11. Transfer 750 ul (or less) of solution to a new sterile 2 ml microtube. (Do not touch the pellet)
12. Add 1200 ul of solution C4 and vortex.
(Solution C4 is a saline solution that allows the DNA to attach to the silica inside the Spin Filter.)
13. Transfer 675 ul of the solution to a spin filter and cetrifuge for 1 minture. Decant the flow through (Remove the liquid in the bottom of the spin filter).
14. Repeat step 13 until no solution is left.
15. Add 500 ul of solution C5 and cetrifuge for 30 seconds. Discard the flow through.
(Solution C5 is an ethanol based wash solution. The ethanol cleans the DNA that is attached to the silica filter of any excess salt, humic acid, and other contaminants.)
16. Centrifuge an additional 30 seconds to remove any excess C5 from the silica filter.
17. Transfer the filter to a labeled sterile 2 ml microtube.
18. Add 30 ul of solution C6 to the membrane filter. Be sure to get the solution onto the actual white membrane in teh filter.
(Solution C6 is a sterile elution buffer (10 mM Tris) that wets the membrane and effectively releases the DNA from the membrane.)
19. Centrifuge for 30 seconds.
20. Discard teh filter and store the tube containing DNA at -20 degrees Celsius.

Currently reading:
http://schloss.micro.umass.edu/mothur/The_effects_of_eutrophication_on_the_structure_of_bacterial_biofilms

Stranger in a Strange Land, Robert A. Heinlein