Streptococcus pneumoniae Genomic DNA - Basic Information

Culture Medium	Peptone: 10.0g, Beef powder: 3.0g, Sodium chloride: 5.0g, Agar: 15.0g, pH: 7.3±0.1 (25℃)
Growth Conditions	37℃; 18-24h; Aerobic;
Storage Conditions	2-8℃
Procedure	Before use, add anhydrous ethanol to buffer GD and rinse solution PW; the volume to be added is indicated on the bottle label. 1. Take 1-5ml of bacterial culture, centrifuge at 10,000rpm (11,500×g) for 1 min, and carefully remove the supernatant. 2. Add 200μL of buffer GA to the bacterial pellet, vortex until the pellet is completely suspended. Note: For gram-positive bacteria that are difficult to lyse, you may skip step 2 and instead use lysozyme for wall-breaking treatment. The specific method is: add 110μL of buffer (20mM Tris, pH8.0; 2mM Na2-EDTA; 1.2% Triton) and 70μL of lysozyme solution (50mg/mL, self-prepared, catalog number: RT401), and incubate at 37℃ for over 30 min. If RNA removal is needed, add 4μL of RNase A (100mg/mL) solution (self-prepared, catalog number: RT405-12), vortex for 15 sec, and let stand at room temperature for 5 min. 3. Add 20μL of Proteinase K solution to the tube and mix well. 4. Add 220μL of buffer GB, vortex for 15 sec, incubate at 70℃ for 10 min until the solution becomes clear, then briefly centrifuge to remove droplets from the inside of the cap. Note: A white precipitate may form when adding buffer GB, but it usually dissolves at 70℃ and will not affect subsequent experiments. If the solution does not become clear, cell lysis may be incomplete, potentially resulting in low DNA yield or impure DNA. 5. Add 220μL of anhydrous ethanol, vortex thoroughly for 15 sec. Flocculent precipitate may appear at this stage; briefly centrifuge to remove droplets from the inside of the cap. 6. Transfer the solution and flocculent precipitate from the previous step into an adsorption column CB3 (placed in a collection tube), centrifuge at 12,000rpm (13,400×g) for 30 sec, discard the waste liquid, and return the adsorption column CB3 to the collection tube. 7. Add 500μL of buffer GD (check beforehand if anhydrous ethanol has been added) to the adsorption column CB3, centrifuge at 12,000rpm (13,400×g) for 30 sec, discard the waste liquid, and return the adsorption column CB3 to the collection tube. 8. Add 600μL of rinse solution PW (check beforehand if anhydrous ethanol has been added) to the adsorption column CB3, centrifuge at 12,000rpm (13,400×g) for 30 sec, discard the waste liquid, and return the adsorption column CB3 to the collection tube. 9. Repeat step 8. 10. Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000rpm (13,400×g) for 2 min, discard the waste liquid. Let the adsorption column CB3 stand at room temperature for several minutes to thoroughly dry any residual rinse solution from the adsorption material. Note: This step aims to remove residual rinse solution, as ethanol remnants may interfere with subsequent enzymatic reactions (such as digestion, PCR, etc.). 11. Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200μL of elution buffer TE dropwise to the center of the adsorption membrane, let stand at room temperature for 2-5 min, then centrifuge at 12,000rpm (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 50μL, as a smaller volume may reduce recovery efficiency. The pH of the elution buffer significantly affects elution efficiency. If using ddH2O as the eluent, ensure its pH is between 7.0-8.5; a pH below 7.0 will reduce elution efficiency. DNA products should be stored at -20℃ to prevent degradation. To increase genomic DNA yield, the centrifuged solution can be reapplied to the adsorption column CB3, let stand at room temperature for 2 min, then centrifuge at 12,000rpm (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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