Klebsiella pneumoniae Genomic DNA - Basic Information

Storage Conditions	2-8℃
Operation Steps	Before use, add anhydrous ethanol to Buffer GD and Washing Solution PW; refer to the labels on the bottles 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 remove the supernatant as thoroughly as possible. 2. Add 200 μL Buffer GA to the bacterial pellet and vortex until the bacteria are completely resuspended. Note: For Gram-positive bacteria with tough cell walls, skip step 2 and use a lysozyme solution for cell wall disruption. The specific method is: 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, self-provided, catalog number: RT401), and treat at 37℃ for over 30 min. If RNA removal is needed, add 4 μL RNase A (100 mg/mL) solution (self-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 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 inside of the tube cap. Note: A white precipitate may form when adding Buffer GB, 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 extraction. 5. Add 220 μL anhydrous ethanol, vortex thoroughly for 15 sec; flocculent precipitate may appear at this stage. Briefly centrifuge to remove water droplets from the inside of the tube cap. 6. Transfer the solution and flocculent precipitate from the previous step into a CB3 adsorption column (place the column in a collection tube), centrifuge at 12,000 rpm (13,400×g) for 30 sec, discard the waste liquid, and place the CB3 column back into the collection tube. 7. Add 500 μL Buffer GD to the CB3 adsorption column (ensure anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (13,400×g) for 30 sec, discard the waste liquid, and place the CB3 column back into the collection tube. 8. Add 600 μL Washing Solution PW to the CB3 adsorption column (ensure anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (13,400×g) for 30 sec, discard the waste liquid, and place the CB3 column back into the collection tube. 9. Repeat step 8. 10. Place the CB3 adsorption column back into the collection tube, centrifuge at 12,000 rpm (13,400×g) for 2 min, and discard the waste liquid. Let the CB3 column stand at room temperature for several minutes to thoroughly dry the residual washing solution from the adsorption material. Note: The purpose of this step is to remove residual washing solution from the column; residual ethanol in the washing solution can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.). 11. Transfer the CB3 adsorption column 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 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; a smaller volume may affect recovery efficiency. The pH of the elution buffer has a significant impact on elution efficiency. If using ddH2O as the eluent, ensure its pH is within the range of 7.0-8.5; a pH below 7.0 will reduce elution efficiency. DNA products should be stored at -20℃ to prevent degradation. To increase the yield of genomic DNA, the centrifuged solution can be added back to the CB3 adsorption column, let stand at room temperature for 2 min, and centrifuged 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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