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Stool gDNA Extraction Kit(SpinColumn)

Packing Specification：

Product Introduction:
In cutting-edge fields such as microbiome research and gut microbiota analysis, accurate extraction of stool genomic DNA is a critical prerequisite for obtaining reliable experimental data. However, complex components in stool samples, such as inhibitory factors including humic acid, polysaccharides, and proteins, often render conventional DNA purification methods ineffective, leading to problems such as PCR amplification failure and low-quality sequencing data, which severely hinder scientific research progress.
This kit can accurately and efficiently remove various inhibitory factors while maximizing the recovery of high-quality genomic DNA. The unique combination of Buffer TL, 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 steps including impurity removal, inhibitor elimination, washing, and centrifugation, PCR inhibitors, cellular metabolites, proteins, and other impurities are removed by the Impurity Remover, Inhibitor Removal Buffer, and Washing Buffer. 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 high-quality 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.
5.Accurately and efficiently removes various inhibitory factors while maximizing the recovery of high-quality genomic DNA.

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

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

1.Collect approximately 200-220 mg of stool into a 2 mL centrifuge tube and place on ice. For frozen samples, do not thaw before adding Buffer TL, otherwise DNA is prone to degradation.

2.Add 1.4 mL of Buffer TL, vortex continuously for 1 minute or until fully mixed. Ensure thorough vortex mixing; otherwise, yield will be significantly reduced.

3.Incubate the resuspension at 70°C for 5 minutes. This heating step can increase DNA yield by 3-5 times and assist in lysing bacteria and parasites. For certain difficult-to-lyse cells (such as Gram-positive bacteria), the temperature can be increased to 95°C.

4.Vortex for 15 seconds, then incubate at room temperature for 1 minute. Centrifuge at maximum speed for 1 minute to pellet stool particles.

5.Transfer 900 μL of the supernatant to a 1.5 mL centrifuge tube, add 100 μL of Impurity Remover Buffer AB, immediately vortex for 1 minute or until fully mixed, and incubate at room temperature for 1 minute. Centrifuge at maximum speed for 3 minutes to remove impurities.

6.Transfer all the supernatant to a 1.5 mL centrifuge tube, centrifuge at maximum speed for 3 minutes.
7.Transfer 210 μL of the supernatant to a 1.5 mL centrifuge tube, add 20 μL of Proteinase K (20mg/mL) solution, mix thoroughly. Add 200 μL of Binding Buffer CB, vortex for 15 seconds to mix well. Incubate at 70℃ for 10 minutes.

8.If the yield is low, transfer more supernatant and proportionally increase the amounts of Proteinase K, Binding Buffer, and isopropanol used in subsequent steps.

9.After cooling, add 100 μL of isopropanol and vortex to mix.

10.Transfer the resulting solution (including any possible precipitation) to a Spin Column AC (placed in a collection tube), centrifuge at 13,000 rpm for 30 seconds, and discard the waste liquid in the collection tube.

11.Add 500 μL of Inhibitor Removal Buffer IR, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

12.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.

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

14.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).

15.Transfer the Spin Column AC to a clean centrifuge tube. Add 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 50 μL (excessively small volumes reduce elution efficiency and DNA yield).

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