Products

Bacteria gDNA Extraction Kit(SpinColumn)

Packing Specification：

Product Introduction:
The unique combination of Binding Buffer and Proteinase K rapidly lyses bacterial cells and inactivates intracellular nucleases. Genomic DNA is then selectively adsorbed onto the silica membrane of the spin column under high chaotropic salt conditions. Through a series of rapid washing and centrifugation steps, Inhibitor Removal Buffer and Wash Buffer remove impurities such as cellular metabolites and proteins. Finally, pure genomic DNA is eluted from the silica membrane using a low-salt Elution Buffer.

Product Features:
1.The silica membrane in the spin column is made of high-quality special adsorption membrane, ensuring minimal variation in adsorption capacity between columns.
2.No need for toxic reagents such as phenol or ethanol precipitation steps.
3.Fast and simple; operation for a single sample can generally be completed within 30 minutes.
4.Multiple column washes ensure high purity. The typical OD260/OD280 ratio is 1.7-1.9, and the DNA length can reach 30 kb - 50 kb, which can be directly used for PCR, Southern blot, and various restriction enzyme digestion reactions.

Application Scope:
Suitable for the rapid extraction of genomic DNA from various bacteria.

Operation Steps:
Note: Before first use, add the specified amount of anhydrous ethanol to Wash Buffer WB, mix thoroughly, and mark the box to indicate ethanol has been added to avoid repeated addition!

1.Take 0.5-2 mL of cultured bacterial broth (up to 2×10⁹ cells), centrifuge at 10,000 rpm for 30 seconds. Aspirate and discard the supernatant as much as possible to collect the bacterial pellet.
The initial processing volume can be adjusted according to bacterial density, cell type, and expected yield. However, the maximum adsorption capacity of the spin column is 20 μg of genomic DNA. Excessive bacterial pellets exceeding the maximum adsorption capacity will severely reduce the yield.

2.Add 200 μL of Buffer RB to resuspend, centrifuge at 10,000 rpm for 30 seconds, and discard the supernatant. Vortex or pipette to fully resuspend the cells in 180 μL of Buffer RB.
Note: For Gram-positive bacteria that are difficult to lyse, skip Step 2 and perform cell wall lysis with lysozyme as follows: Add 180 μL of buffer (20 mM Tris, pH 8.0; 2 mM Na₂-EDTA; 1.2% Triton X-100; add lysozyme to a final concentration of 20 mg/mL immediately before use—lysozyme must be prepared by dissolving lysozyme powder in the buffer, otherwise it will be inactive), and incubate at 37℃ for more than 30 minutes.

3.Add 20 μL of Proteinase K (20 mg/mL) solution, mix thoroughly. Add 200 μL of Binding Buffer CB, immediately vortex to mix well, and incubate at 70℃ for 10 minutes.
Optional step: If significant RNA contamination is present, add 4 μL of RNase A (100 mg/mL) solution before adding 200 μL of Binding Buffer CB, vortex to mix, and incubate at room temperature for 5-10 minutes.

4.After cooling, add 100 μL of isopropanol and immediately vortex to mix thoroughly (flocculent precipitation may occur).
Immediate vortexing or pipetting to mix thoroughly is critical in this step. Inadequate mixing will severely reduce yield. For viscous samples that are difficult to mix, vortex for 15 seconds if necessary.

5.Load the mixture (including any precipitation) into a Spin AC column (placed in a collection tube). Centrifuge at 13,000 rpm for 30-60 seconds and discard the waste liquid in the collection tube.
6.Add 500 μL of Inhibitor Removal Buffer IR, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

7.Add 600 μL of Wash Buffer WB (ensure anhydrous ethanol has been added!), centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

8.Add another 600 μL of Wash Buffer WB, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

9.Place the Spin AC column back into the empty collection tube and centrifuge at 13,000 rpm for 2 minutes to thoroughly remove residual Wash Buffer (residual ethanol may inhibit downstream reactions).

10.Remove the Spin AC column and place it into a clean centrifuge tube. Add 100 μL of Elution Buffer EB (preheating the elution buffer in a 65-70℃ water bath beforehand improves elution efficiency) to the center of the adsorption membrane. Incubate at room temperature for 3-5 minutes, then centrifuge at 12,000 rpm for 1 minute. Transfer the eluted solution back into the spin column, incubate at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute.
Note: Larger elution volumes result in higher elution efficiency. For higher DNA concentration, reduce the elution volume (minimum 30 μL; volumes smaller than this reduce elution efficiency and DNA yield).
Store DNA at 2-8℃ for short-term use or -20℃ for long-term storage.