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Week 2Session2

Week 2Session2 - Bi 1x Spring 2010 Week 2 Session 2 DNA...

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Page 1 of 6 Bi 1x, Spring 2010 Week 2, Session 2 DNA extraction Spectrophotometry of DNA DNA Extraction In this component of today’s lab, you will extract the genomic DNA from the microbes in the environmental samples you collected from the Caltech ponds using a specialized extraction kit. Materials (per person): PowerBead tube containing your sample from the night before Extraction solution set – C1, C2, C3, C4, C5, C6 Four 2 ml collection tubes Spin filter in collection tube Protocol 1. Ensure the PowerBead tube containing your sample is fully thawed. The PowerBead Tube contains a bead matrix that will help physically lyse your sample as well as a buffer that will (a) help disperse the soil particles, (b) begin to dissolve humic acids (contaminants that occur in environmental samples) and (c) protect nucleic acids from degradation. 2. Gently vortex to mix (vortex on ~50% power for 3-4 sec). Gentle vortexing mixes the components in the PowerBead Tube and begins to disperse the sample in the PowerBead Solution. 3. Add 60 µ l of Solution C1 and invert the tube several times or vortex briefly. Solution 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. 4. Secure PowerBead Tubes horizontally in the Vortex Adapter tube holder (ask your TA for help). Vortex at maximum speed for 10 minutes. The vortexing step is critical for complete homogenization and cell lysis. Cells are lysed by a combination of chemical agents from steps 1-3 and mechanical shaking introduced at this step. By randomly shaking the beads in the presence of disruption agents, collision of the beads with microbial cells will cause the cells to break open.
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Page 2 of 6 5. Centrifuge tubes at 10,000 x g in a microcentrifuge for 30 seconds at room temperature. CAUTION: Make sure the PowerBead Tubes rotate freely in your centrifuge without rubbing. Be sure not to exceed 10,000 x g or tubes may break. This step will pellet the lysis beads and cell debris to allow them to be separated from your sample. 6. Transfer the supernatant (the upper, non-pelleted liquid phase) to a clean 2 ml Collection Tube. Expect between 400 to 500 µ l of supernatant at this step. The exact recovered volume depends on the absorbancy of your starting material and is not critical for the procedure to be effective. The supernatant may be dark in appearance and still contain some environmental debris. Subsequent steps in the protocol will remove these contaminants.
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