E. coli Genomic DNA - Basic Information

Storage Conditions	2-8°C
Procedure	Before use, add absolute ethanol to Buffer GD and Wash Solution PW, referring to the bottle labels for the volume to add. 1. Take 1-5 ml of bacterial culture, centrifuge at 10,000 rpm (11,500×g) for 1 min, and carefully remove the supernatant. 2. Add 200 μL of Buffer GA to the bacterial pellet and vortex until the pellet is completely resuspended. Note: For Gram-positive bacteria with tough cell walls, skip step 2 and perform cell wall digestion with lysozyme solution. Specifically, add 110 μL of buffer (20 mM Tris, pH 8.0; 2 mM Na2-EDTA; 1.2% Triton) and 70 μL of lysozyme solution (50 mg/mL, prepared by user, catalog number: RT401), and incubate at 37°C for over 30 min. If RNA removal is needed, add 4 μL of RNase A solution (100 mg/mL, prepared by user, catalog number: RT405-12), vortex for 15 sec, and let stand at room temperature for 5 min. 3. Add 20 μL of Proteinase K solution to the tube and mix well. 4. Add 220 μL of Buffer GB, vortex for 15 sec, incubate at 70°C for 10 min until the solution becomes clear, and briefly centrifuge to remove droplets from the cap. Note: A white precipitate may form when Buffer GB is added, which typically disappears at 70°C and does not affect subsequent steps. If the solution does not become clear, cell lysis is incomplete, which may result in low DNA yield or impure DNA. 5. Add 220 μL of absolute ethanol, vortex thoroughly for 15 sec until a flocculent precipitate may appear, and briefly centrifuge to remove droplets from the cap. 6. Transfer the solution and any 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 column back in the collection tube. 7. Add 500 μL of Buffer GD to Spin Column CB3 (ensure absolute ethanol has been added before use), centrifuge at 12,000 rpm (13,400×g) for 30 sec, discard the flow-through, and place the column back in the collection tube. 8. Add 600 μL of Wash Solution PW to Spin Column CB3 (ensure absolute ethanol has been added before use), centrifuge at 12,000 rpm (13,400×g) for 30 sec, discard the flow-through, and place the column back in the collection tube. 9. Repeat step 8. 10. Return Spin Column CB3 to the collection tube, centrifuge at 12,000 rpm (13,400×g) for 2 min, and discard the flow-through. Let the column stand at room temperature for a few minutes to completely dry the residual wash solution from the membrane. Note: This step removes residual wash solution, as ethanol in the wash solution can interfere with downstream enzymatic reactions (e.g., restriction digestion, PCR). 11. Transfer Spin Column CB3 into a clean centrifuge tube, add 50-200 μL of Elution Buffer TE dropwise to the center of the membrane, let stand at room temperature for 2-5 min, centrifuge at 12,000 rpm (13,400×g) for 2 min, and 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 elution efficiency. If using ddH2O as the eluent, ensure its pH is between 7.0 and 8.5; a pH below 7.0 reduces elution efficiency. DNA products should be stored at -20°C to prevent degradation. To increase genomic DNA yield, reload the collected solution onto Spin Column CB3, let stand at room temperature for 2 min, and centrifuge at 12,000 rpm (13,400×g) for 2 min.
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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