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Page 1 of 6 Bi 1x, Spring 2009 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 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.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 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. 7. Add 250 l of Solution C2 and vortex for 5 seconds. Incubate at 4°C for 5 minutes. Solution C2 contains a reagent to precipitate non-DNA organic and inorganic material including humic substances, cell debris, and proteins. It is important to remove contaminating organic and inorganic matter that may reduce DNA purity and inhibit downstream DNA applications, like PCR. 8. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g. 9. Avoiding the pellet, transfer up to 600 l of supernatant to a clean 2 ml Collection Tube (provided). The pellet at this point contains non-DNA organic and inorganic material including humic acid, cell debris, and proteins. For the best DNA yields, and quality, avoid transferring any of the pellet. 10. Add 200 l of Solution C3 and vortex briefly. Incubate at 4°C for 5 minutes. Solution C3 is a second reagent to precipitate additional non-DNA organic and inorganic material including humic acid, cell debris, and proteins. 11. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g. 12. Transfer up to 750 l of supernatant to a clean 2 ml Collection Tube. The pellet at this point contains additional non-DNA organic and inorganic material including humic acid, cell debris, and proteins. For the best DNA yields, and quality, avoid transferring any of the pellet. 13. Add 1.2ml of Solution C4 to the supernatant (be careful solution doesn’t exceed rim of tube) and vortex for 5 seconds.Page 3 of 6 Solution C4 is a high concentration salt solution. DNA binds tightly to silica at high salt concentrations, and this buffer will adjust the DNA solution salt concentrations to allow binding of DNA (but not non-DNA organic and inorganic material that may still be present at low levels) to the Spin Filters. 14. Load approximately 675 l of your sample onto a Spin Filter and centrifuge at 10,000 x g for 1 minute at room temperature. Discard the flow through and add an additional 675 l of supernatant to the Spin Filter and centrifuge at 10,000 x g for 1 minute at room temperature. Load the remaining supernatant onto the Spin Filter and centrifuge at 10,000 x g for 1 minute at room temperature. Note: A total of three loads for each sample processed are required. DNA is selectively bound to the silica membrane in the Spin Filter device in the high salt solution. Contaminants pass through the filter membrane, leaving only DNA bound to the membrane. 15. Add 500 l of Solution C5 and centrifuge at room temperature for 30 seconds at 10,000 x g. Solution C5 is an ethanol based wash solution used to further clean the DNA that is bound to the silica filter membrane in the Spin Filter. This wash solution removes residual salt, humic acid, and other contaminants while allowing the DNA to stay bound to the silica membrane. 16. Discard the flow through from the 2 ml Collection tube. This flow through fraction is just non-DNA organic and inorganic waste removed from the silica Spin Filter membrane by the ethanol wash solution. 17. Centrifuge at room temperature for 1 minute at 10,000 x g. This second spin removes residual Solution C5 (ethanol wash solution). It is critical to remove all traces of wash solution because the ethanol in Solution C5 can interfere with many downstream DNA applications such as PCR, restriction digests, and gel electrophoresis. 18. Carefully place Spin Filter in a clean 2 ml Collection Tube. Avoid splashing any Solution C5 onto the Spin Filter. It is important to avoid any traces of the ethanol-based wash solution. 19. Add 70 l of Solution C6 to the center of the white filter membrane. Placing the Solution C6 (sterile elution buffer) in the center of the small white membrane will make sure the


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