Products

Sputum gDNA Extraction Kit(SpinColumn)

Packing Specification：

Application Scope:
Suitable for rapid extraction of genomic DNA from sputum samples.

Product Introduction:
Sputum is viscous, large in volume, and cells are relatively scattered, making it unsuitable for direct genomic DNA extraction. Sputum needs to be liquefied before nucleic acid extraction and purification to release bacterial DNA from the sputum. Conventional liquefaction with NaOH solution can completely liquefy sputum, but it is highly alkaline, which easily denatures and unwinds both host and bacterial DNA, leading to severe DNA degradation that cannot be extracted and purified by traditional spin column methods. Through extensive testing and verification, this kit can simply and efficiently liquefy sputum, release microorganisms such as bacteria from the sputum. The unique combination of Binding Buffer CB 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 special adsorption membranes, with minimal variation in adsorption capacity between columns and good reproducibility, overcoming the drawback of unstable membrane quality in domestic kits.
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 45-60 minutes.
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.

Operation Steps:
Pre-Experiment Preparation:
1.Preheat the water bath to 55℃ in advance.

2.Ensure the specified amount of absolute ethanol has been added to the Washing Buffer WB bottle, mix thoroughly. After addition, mark the corresponding box to indicate ethanol has been added to avoid repeated additions.

3.Pretreat the adsorption column with Equilibration Buffer as a mandatory step; refer to "About the Use of Equilibration Buffer" above for specific methods.

Operation Steps:
1.Take 400 μL of fresh or thawed sputum sample, add it to a 1.5 mL centrifuge tube containing 40 μL of Proteinase K solution (20mg/mL), and mix by pipetting with a large-bore tip.

2.Incubate the lysate in a 55℃ water bath for 3 hours or overnight until the viscous sputum is completely digested, gently vortexing several times during incubation to assist lysis.
Optional step: If RNA contamination is significant, add 20 μL of RNase A (25mg/mL, prepared by user) solution after Step 2, vortex to mix, and incubate at room temperature for 5-10 minutes to remove RNA.

3.Add 400 μL of Binding Buffer CB, immediately vortex thoroughly or mix by pipetting, then incubate at 70℃ for 10 minutes.

4.After the centrifuge tube cools down, add 200 μL of isopropanol, immediately vortex thoroughly to mix. Flocculent precipitation may occur at this step.

5.Use a 1 mL tip to aspirate no more than 700 μL of the mixture each time, transfer it to a Spin Column AC (placed in a collection tube), centrifuge at 13,000 rpm for 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 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 100 μL of Elution Buffer TE (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.

11.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 50 μL (excessively small volumes reduce elution efficiency and DNA yield).

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