E. coli Genomic DNA - Basic Information

Medium	Peptone: 10.0g, Beef extract: 3.0g, Sodium chloride: 5.0g, Agar: 15.0g, pH: 7.3±0.1 (25°C)
Growth Conditions	37°C; 18-24h; Aerobic
Growth Characteristics	On nutrient agar medium at 37°C with 3 days of cultivation, the bacteria are rod-shaped, 0.61μm × 1.22~2.13μm, Gram-negative, non-spore-forming.
Storage Conditions	2-8°C
Morphology	Colony diameter 1-2mm, round, irregular edge, opaque, pale yellow front, slightly raised center, smooth and glossy surface, moist texture, easily picked, Gram-negative (red), rod-shaped, pure.
Procedure	Before use, add absolute ethanol to Buffer GD and Wash Buffer PW as per the bottle label. 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 pellet, vortex until thoroughly suspended. Note: For difficult-to-lyse Gram-positive bacteria, skip step 2 and add lysozyme solution for cell wall digestion as follows: add 110 μL buffer (20mM Tris, pH8.0; 2mM Na2-EDTA; 1.2% Triton) and 70 μL lysozyme solution (50 mg/mL, user-provided, catalog number: RT401), treat at 37°C for over 30 min. To remove RNA, add 4 μL RNase A (100 mg/mL) solution (user-provided, catalog number: RT405-12), vortex for 15 sec, and let stand at room temperature for 5 min. 3. Add 20 μL Proteinase K solution, mix well. 4. Add 220 μL Buffer GB, vortex for 15 sec, and incubate at 70°C for 10 min until the solution becomes clear. Briefly centrifuge to remove droplets from the cap. Note: A white precipitate may form upon adding Buffer GB, which typically disappears at 70°C and does not affect subsequent experiments. If the solution does not clear, cell lysis is incomplete, which may result in low yield and impure DNA. 5. Add 220 μL absolute ethanol, vortex thoroughly for 15 sec; a flocculent precipitate may appear. Briefly centrifuge to remove droplets from the cap. 6. Transfer the solution and precipitate to Adsorption 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 column back into the collection tube. 7. Add 500 μL Buffer GD (ensure ethanol has been added) to Adsorption Column CB3, centrifuge at 12,000 rpm (13,400×g) for 30 sec, discard the flow-through, and place the column back. 8. Add 600 μL Wash Buffer PW (ensure ethanol has been added) to Adsorption Column CB3, centrifuge at 12,000 rpm (13,400×g) for 30 sec, discard the flow-through, and place the column back. 9. Repeat step 8. 10. Place Adsorption Column CB3 back into the collection tube, centrifuge at 12,000 rpm (13,400×g) for 2 min, discard the flow-through, and let the column air-dry at room temperature for a few minutes to thoroughly remove residual wash buffer. Note: This step ensures removal of residual ethanol in the wash buffer, as ethanol can interfere with downstream enzymatic reactions (e.g., digestion, PCR). 11. Transfer Adsorption Column CB3 to a clean centrifuge tube, carefully add 50-200 μL Elution Buffer TE to the center of the membrane, let stand for 2-5 min at room temperature, then centrifuge at 12,000 rpm (13,400×g) for 2 min to collect the solution. Note: The elution buffer volume should not be less than 50 μL, as smaller volumes reduce recovery efficiency. The pH of the elution buffer significantly affects efficiency; if using ddH2O, ensure pH is between 7.0-8.5, as pH below 7.0 lowers recovery. Store DNA at -20°C to prevent degradation. To increase yield, the collected solution can be reapplied to Adsorption Column CB3, let stand for 2 min, and centrifuged again at 12,000 rpm (13,400×g) for 2 min.
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📋 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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