Klebsiella pneumoniae Genomic DNA - Basic Information

Storage Conditions	2-8℃
Operation Steps	Before use, please add absolute ethanol to Buffer GD and Wash Buffer PW, refer to the label on the bottle for the volume to add; 1. Take 1-5 ml of bacterial culture, centrifuge at 10,000 rpm (11,500×g) for 1 minute, and remove the supernatant as much as possible; 2. Add 200 μL Buffer GA to the bacterial pellet, vortex until the bacteria are completely resuspended. Note: For Gram-positive bacteria with hard-to-break cell walls, skip step 2 and add lysozyme solution for cell wall disruption. The specific method is: add 110 μL buffer (20mM Tris, pH 8.0; 2mM Na2-EDTA; 1.2% Triton), and 70 μL lysozyme solution (50 mg/mL, customer-provided, catalog number: RT401), treat at 37℃ for over 30 minutes. If RNA removal is needed, add 4 μL RNase A (100 mg/mL) solution (customer-provided, catalog number: RT405-12), vortex for 15 seconds, and let stand at room temperature for 5 minutes; 3. Add 20 μL Proteinase K solution to the tube, mix well; 4. Add 220 μL Buffer GB, vortex for 15 seconds, place at 70℃ for 10 minutes; the solution should become clear. Briefly centrifuge to remove droplets from the inner wall of the tube cap. Note: When adding Buffer GB, a white precipitate may form, which usually disappears at 70℃ and does 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 DNA; 5. Add 220 μL absolute ethanol, vortex thoroughly for 15 seconds; flocculent precipitate may appear at this point. Briefly centrifuge to remove droplets from the inner wall of the tube cap; 6. Transfer the solution and flocculent precipitate from the previous step into an adsorption column CB3 (place the column in a collection tube), centrifuge at 12,000 rpm (13,400×g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 back into the collection tube; 7. Add 500 μL Buffer GD to the adsorption column CB3 (please check if absolute ethanol has been added before use), centrifuge at 12,000 rpm (13,400×g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 back into the collection tube; 8. Add 600 μL Wash Buffer PW to the adsorption column CB3 (please check if absolute ethanol has been added before use), centrifuge at 12,000 rpm (13,400×g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 back into the collection tube; 9. Repeat operation step 8; 10. Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm (13,400×g) for 2 minutes, discard the waste liquid. Let the adsorption column CB3 stand at room temperature for several minutes to completely dry any residual wash buffer in the adsorption material. Note: This step aims to remove residual wash buffer from the adsorption column, as residual ethanol in the wash buffer can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.); 11. Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200 μL Elution Buffer TE dropwise to the center of the adsorption membrane, let stand at room temperature for 2-5 minutes, centrifuge at 12,000 rpm (13,400×g) for 2 minutes, and collect the solution into the centrifuge tube. Note: The volume of elution buffer should not be less than 50 μL, as too small a volume affects recovery efficiency. The pH value of the elution buffer significantly impacts elution efficiency. If using ddH2O as the eluent, ensure its pH is between 7.0-8.5; a pH below 7.0 will reduce elution efficiency; and DNA products should be stored at -20℃ to prevent DNA degradation. To increase the yield of genomic DNA, the centrifuged solution can be added back to the adsorption column CB3, let stand at room temperature for 2 minutes, and centrifuge at 12,000 rpm (13,400×g) for 2 minutes;
Sharing Method	Non-profit 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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