Genomic DNA from Klebsiella aerogenes Tindall et al. - Basic Information

Storage Conditions	2-8°C
Procedure	Before use, please add anhydrous ethanol to Buffer GD and Wash Buffer PW. Refer to the labels on the bottles for the volumes 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 completely. 2. Add 200 μL Buffer GA to the bacterial pellet and vortex until the cells are thoroughly resuspended. Note: For Gram-positive bacteria that are difficult to lyse, you may skip step 2 and perform lysozyme treatment as follows: add 110 μL of buffer (20 mM Tris, pH 8.0; 2 mM Na₂-EDTA; 1.2% Triton) and 70 μL lysozyme solution (50 mg/mL, user-provided, Cat. No. RT401), incubate at 37°C for at least 30 min. If RNA removal is needed, add 4 μL RNase A (100 mg/mL) solution (user-provided, Cat. No. RT405-12), vortex for 15 sec, and leave 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, and incubate at 70°C for 10 min. The solution should become clear. Briefly centrifuge to remove droplets from the lid. Note: A white precipitate may appear upon addition of Buffer GB, but it usually disappears during the 70°C incubation and does not affect the experiment. If the solution does not become clear, cell lysis may be incomplete, which could reduce DNA yield and purity. 5. Add 220 μL anhydrous ethanol and vortex thoroughly for 15 sec. A flocculent precipitate may appear. Briefly centrifuge to remove droplets from the lid. 6. Transfer the solution and any precipitate from the previous step into an 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 to the adsorption column CB3 (ensure anhydrous 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 into the collection tube. 8. Add 600 μL Wash Buffer PW to the adsorption column CB3 (ensure anhydrous 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 into the collection tube. 9. Repeat step 8. 10. Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm (13,400 × g) for 2 min, and discard the flow-through. Then leave the column at room temperature for several minutes to completely dry the residual wash buffer from the membrane. Note: This step removes residual ethanol from the wash buffer, which can interfere with downstream enzymatic reactions (e.g., digestion, PCR). 11. Transfer the adsorption column CB3 to a clean centrifuge tube. Add 50-200 μL Elution Buffer TE to the center of the membrane, let stand at room temperature for 2-5 min, and centrifuge at 12,000 rpm (13,400 × g) for 2 min to collect the DNA solution. Note: The elution 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 ddH₂O is used as the eluent, ensure its pH is within 7.0-8.5; pH below 7.0 reduces elution efficiency. Store the DNA product at -20°C to prevent degradation. To increase genomic DNA yield, you may reload the eluted solution onto the same column, let stand at room temperature for 2 min, and centrifuge again 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.