Mycobacterium tuberculosis Genomic DNA - Basic Information

Culture Medium	Peptone: 10.0g, Beef powder: 3.0g, Sodium chloride: 5.0g, Agar: 15.0g, pH value: 7.3±0.1 (25°C)
Growth Conditions	37°C; 18-24h; Aerobic;
Storage Conditions	2-8°C
Operation Steps	Before use, add anhydrous ethanol to buffer GD and rinse PW, and refer to the label on the bottle for the volume to be added; 1. Take 1-5ml of bacterial culture solution, centrifuge at 10,000rpm (11,500×g) for 1min, and remove the supernatant as much as possible; 2. Add 200μL of buffer GA to the bacterial pellet, and vortex until the pellet is completely suspended. Note: For Gram-positive bacteria with difficult cell walls, skip step 2 and use lysozyme solution for cell wall digestion. The specific method is: add 110μL of buffer (20mM Tris, pH8.0; 2mM Na2-EDTA; 1.2% Triton), and 70μL of lysozyme solution (50mg/mL, customer-provided, catalog number: RT401), and treat at 37°C for more than 30min. If RNA removal is required, add 4μL of RNase A (100mg/mL) solution (customer-provided, catalog number: RT405-12), vortex for 15sec, and let it stand at room temperature for 5min; 3. Add 20μL of Proteinase K solution to the tube, and mix well; 4. Add 220μL of buffer GB, vortex for 15sec, and place at 70°C for 10min. The solution should become clear. Briefly centrifuge to remove water droplets from the inner wall of the tube cap. Note: White precipitate may form when buffer GB is added, which usually disappears during the 70°C incubation 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 220μL of anhydrous ethanol, and vortex thoroughly for 15sec. Flocculent precipitate may appear at this stage. Briefly centrifuge to remove water droplets from the inner wall of the tube cap; 6. Transfer the solution and flocculent precipitate obtained from the previous step into an adsorption column CB3 (place the adsorption column in a collection tube), centrifuge at 12,000rpm (13,400×g) for 30sec, discard the waste liquid, and place the adsorption column CB3 back into the collection tube; 7. Add 500μL of buffer GD to the adsorption column CB3 (check if anhydrous ethanol has been added before use), centrifuge at 12,000rpm (13,400×g) for 30sec, discard the waste liquid, and place the adsorption column CB3 back into the collection tube; 8. Add 600μL of rinse PW to the adsorption column CB3 (check if anhydrous ethanol has been added before use), centrifuge at 12,000rpm (13,400×g) for 30sec, discard the waste liquid, and place the adsorption column CB3 back into the collection tube; 9. Repeat step 8; 10. Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000rpm (13,400×g) for 2min, and discard the waste liquid. Leave the adsorption column CB3 at room temperature for several minutes to thoroughly dry the residual rinse solution in the adsorption material. Note: The purpose of this step is to remove residual rinse solution from the adsorption column, as ethanol residue in the rinse solution can affect subsequent enzyme reactions (such as enzymatic digestion, PCR, etc.); 11. Transfer the adsorption column CB3 into a clean centrifuge tube, and add 50-200μL of elution buffer TE dropwise to the center of the adsorption membrane. Let it stand at room temperature for 2-5min, then centrifuge at 12,000rpm (13,400×g) for 2min, and collect the solution into the centrifuge tube. Note: The volume of elution buffer should not be less than 50μL, as a smaller volume will affect recovery efficiency. The pH value of the elution buffer significantly impacts elution efficiency. If using ddH2O as the eluent, ensure its pH value is within the range of 7.0-8.5, as a pH value below 7.0 will reduce elution efficiency; and the DNA product should be stored at -20°C to prevent DNA degradation. To increase the yield of genomic DNA, the centrifuged solution can be added back into the adsorption column CB3, let stand at room temperature for 2min, and then centrifuged at 12,000rpm (13,400×g) for 2min;
Sharing Method	Public welfare sharing

📋 Notes:

1. Storage Conditions:Lyophilized powder should be stored at 2–8°C away from light. After reconstitution or for solution-type products, store at –20°C and avoid repeated freeze-thaw cycles. For short-term frequent use (within 1 week), temporary storage at 4°C is acceptable.
2. Reconstitution Method:Prior to use, briefly centrifuge the lyophilized powder (e.g., 12,000 rpm for 1 minute) to collect the DNA at the bottom of the tube. It is recommended to dissolve in sterile TE buffer (pH 8.0) or sterile ddH₂O (pH 7.0–8.5). Mix gently; avoid vigorous vortexing.
3. Contamination Prevention:Gloves must be worn during operation. Use sterile, nuclease-free consumables. It is recommended to aliquot the reconstituted DNA into single-use portions before freezing, to prevent degradation caused by repeated freeze-thaw cycles and to avoid nuclease contamination.
4. Experimental Suitability:This product is suitable for common molecular biology applications such as PCR, qPCR, restriction enzyme digestion, and library construction. If the downstream assay is sensitive to inhibitors, ensure that the DNA solution is free of residual ethanol or high-concentration salt interference.
5. Intended Use Statement:The product is for research use only and is not intended for clinical diagnostic purposes.

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