Clostridium sporogenes Genomic DNA - Basic Information

Recommended Medium	Peptone: 10.0g, Beef extract powder: 3.0g, Sodium chloride: 5.0g, Agar: 15.0g, pH: 7.3±0.1 (25℃)
Growth Conditions	37℃; 18-24h; Aerobic;
Storage Conditions	2-8℃
Procedure	Before use, add anhydrous ethanol to Buffer GD and Wash Buffer PW. Refer to the label on the bottle for the volume to be added; 1. Take 1-5 ml of bacterial culture, centrifuge at 10,000 rpm (11,500×g) for 1 min, and remove the supernatant as completely as possible; 2. Add 200 μL Buffer GA to the bacterial pellet and vortex until the pellet is thoroughly suspended. Note: For Gram-positive bacteria with tough cell walls, step 2 may be skipped and lysozyme treatment may be performed instead. The specific method is as follows: add 110 μL buffer (20 mM Tris, pH 8.0; 2 mM Na2-EDTA; 1.2% Triton) and 70 μL lysozyme solution (50 mg/mL, prepared by the user, Cat. No.: RT401), and incubate at 37℃ for at least 30 min. If RNA removal is required, add 4 μL RNase A (100 mg/mL) solution (prepared by the user, Cat. No.: RT405-12), vortex for 15 sec, and let stand at room temperature for 5 min; 3. Add 20 μL Proteinase K solution to the tube and mix well; 4. Add 220 μL Buffer GB, vortex for 15 sec, incubate at 70℃ for 10 min. The solution should become clear. Briefly centrifuge to remove water droplets from the inner wall of the tube cap. Note: A white precipitate may form when Buffer GB is added; it usually disappears upon incubation at 70℃ and will not affect subsequent experiments. If the solution does not become clear, it indicates incomplete cell lysis, which may result in low DNA yield and impure extracted DNA; 5. Add 220 μL anhydrous ethanol and mix thoroughly by vortexing for 15 sec. A flocculent precipitate may appear at this point. Briefly centrifuge to remove water droplets from the inner wall of the tube cap; 6. Transfer the solution and flocculent precipitate from the previous step into a Spin Column CB3 (placed in a collection tube). Centrifuge at 12,000 rpm (13,400×g) for 30 sec. Discard the flow-through and place the Spin Column CB3 back into the collection tube; 7. Add 500 μL Buffer GD to Spin Column CB3 (ensure anhydrous ethanol has been added before use). Centrifuge at 12,000 rpm (13,400×g) for 30 sec. Discard the flow-through and place the Spin Column CB3 back into the collection tube; 8. Add 600 μL Wash Buffer PW to Spin Column CB3 (ensure anhydrous ethanol has been added before use). Centrifuge at 12,000 rpm (13,400×g) for 30 sec. Discard the flow-through and place the Spin Column CB3 back into the collection tube; 9. Repeat step 8; 10. Place Spin Column CB3 back into the collection tube and centrifuge at 12,000 rpm (13,400×g) for 2 min. Discard the flow-through. Let Spin Column CB3 stand at room temperature for several minutes to thoroughly air-dry any residual wash buffer in the adsorption material. Note: The purpose of this step is to remove residual wash buffer from the spin column. Residual ethanol in the wash buffer may affect subsequent enzymatic reactions (enzyme digestion, PCR, etc.); 11. Transfer Spin Column CB3 into a clean centrifuge tube. Add 50-200 μL Elution Buffer TE dropwise to the center of the adsorption membrane without touching it. Let stand at room temperature for 2-5 min. Centrifuge at 12,000 rpm (13,400×g) for 2 min and collect the solution in the centrifuge tube. Note: The volume of elution buffer should not be less than 50 μL; too small a volume will reduce recovery efficiency. The pH of the elution buffer has a significant impact on elution efficiency. If ddH2O is used as the eluent, ensure its pH is within the range of 7.0-8.5; a pH below 7.0 will reduce elution efficiency; The DNA product should be stored at -20℃ to prevent degradation. To increase the yield of genomic DNA, the collected solution can be added back to Spin Column CB3, let stand at room temperature for 2 min, and centrifuge at 12,000 rpm (13,400×g) for 2 min;
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