Products

S.aureus gDNA Extraction Kit(SpinColumn)

Packing Specification：

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

Product Features:
1.The silica matrix membranes in the spin columns are all imported special adsorption membranes, with minimal variation in adsorption capacity between columns.
2.No toxic reagents such as phenol are required, nor are steps like ethanol precipitation.
3.Fast and simple: operation for a single sample can usually be completed within 30 minutes.
4.Multiple column washing steps ensure high purity. The typical OD260/OD280 ratio ranges from 1.7 to 1.9, and the DNA length can reach 30kb - 50kb, which can be directly used for PCR, Southern-blot, and various restriction enzyme digestion reactions.

Application Scope:
Suitable for rapid extraction of Staphylococcus aureus genomic DNA.

Operation Steps:
Tips:
Before first use, add the specified amount of absolute ethanol to Washing Buffer WB, mix thoroughly. After addition, mark the corresponding box to indicate ethanol has been added to avoid repeated additions!
Before first use, add 500 μL of Resuscitation Buffer to 250 mg of Staphylococcal Lysozyme, invert to mix until dissolved.

1.Take 0.5-2 mL of cultured bacterial broth (maximum 2×109 cells), centrifuge at 10,000 rpm for 30 seconds, aspirate and discard the supernatant as much as possible, and collect the bacterial pellet. Alternatively, scrape an appropriate amount (about the size of half a mung bean) of bacterial sample.
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 significantly reduce the yield.

2.Add 190 μL of Buffer RB, pipette to mix and resuspend the bacterial pellet. Then add 10 μL of Staphylococcal Lysozyme solution, pipette to mix, and incubate at room temperature for 10 minutes, vortexing once during incubation to ensure complete lysis.

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

4.After cooling, add 100 μL of isopropanol, immediately vortex thoroughly to mix. Flocculent precipitation may occur at this step.
Immediate vortexing or pipetting to mix thoroughly is critical in the above steps. Inadequate mixing will significantly reduce yield. If the sample is viscous and difficult to mix, vortex for 15 seconds if necessary.

5.Transfer the mixture (including any precipitation) to a Spin Column AC (placed in a collection tube), centrifuge at 13,000 rpm for 1 minute, 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 Washing Buffer WB (check if absolute ethanol has been added!), centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

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

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

10.Transfer the Spin Column AC to a clean centrifuge tube. Add 50-100 μL of Elution Buffer EB (pre-warming 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.
Larger elution volumes result in higher elution efficiency. If higher DNA concentration is required, the elution volume can be appropriately reduced, but the minimum volume should not be less than 30 μL (excessively small volumes reduce elution efficiency and DNA yield).

11.DNA can be stored at 2-8℃ for short-term use, or at -20℃ for long-term storage.