Stool DNA Kit

This kit utilizes reversible nucleic acid adsorption properties of glass fiber membranes, can quickly and reliably isolate high quality intact genomic DNA from 100-200 mg fresh or frozen stool samples. Combined with a unique lysis buffer system, it can effectively eliminate humic acid, polysaccharide, phenolic compounds and enzyme inhibitors in stool samples. The purified DNA is suitable for downstream molecular biology techniques such as PCR, enzyme digestion and hybridization.

Proteinase K and RNase A were transported in wet ice packs and stored at -20℃; Other reagents are transported at room temperature, stored at room temperature, valid for 12 months.

1. Please prepare a 70℃ water bath or heating block in advance.

2. If the reagent precipitates, dissolve it at 37℃, and use it after returning to room temperature.

3. Before use, add 30 mL isopropyl alcohol to Buffer SGB, add 18 mL anhydrous ethanol to Buffer SGD, and add 49 mL anhydrous ethanol to Buffer PW.

1. Weigh 100-200 mg stool sample in a 2 mL centrifuge tube and place the tube on ice (If the sample is liquid, pipet 200 µL into the centrifuge tube);

2. Add 600 μL Buffer SLA, 15 μL Proteinase K and 0.25 g grinding beads (1 mm) into the centrifugal tube. Recommend using tissue grinder (KZ-III-F) to grind (60 Hz, 4℃, run 30 s, pause 10 s, grind 5 times) until homogenized, or use a vortex instrument to intermittently shake for 1 min;

3. Incubate at 70℃ for 15 min. Vortex the sample 2-3 times during incubation (If DNA is isolated from gram-positive bacteria, the incubation temperature can be increased to 95℃);

4. Centrifuge at room temperature at 12,000 rpm (~13,400×g) for 10 min, pipet the supernatant into a new 1.5 mL centrifuge tube, taking care not to aspirate precipitate;

5. Add 10 μL RNase A to the supernatant obtained in the previous step, mix the sample throughly by voretxing, incubate at room temperature for 5 min, then add 0.3 times the supernatant volume of Buffer SLB into the centrifuge tube, mix the sample throughly by voretxing and ice bath for 5 min;

6. Centrifuge at room temperature at 12,000 rpm (~13,400×g) for 5 min, pipet the supernatant into a new 1.5 mL centrifuge tube, taking care not to aspirate precipitate;

7. Add equal volume Buffer SGB to the centrifuge tube (make sure that isopropyl alcohol has been added before use), and use pipette to blow and mix well (precipitation may occur);

8. Transfer all of the mixture from previous step into the DNA Spin Column (the amount of each addition should not exceed 600 µL, if more than 600 µL can be added in batches), centrifuge at room temperature at 12,000 rpm (~13,400×g) for 1 min. Discard the filtrate and put the DNA Spin Column into the collection tube;

9. Add 500 μL Buffer SGD (make sure that anhydrous ethanol has been added before use) into the DNA Spin Column, centrifuge at room temperature at 12,000 rpm (~13,400 ×g) for 1 min. Discard the filtrate and put the DNA Spin Column into the Collection tube;

10. Add 600 μL Buffer PW to the DNA Spin Column (make sure that anhydric ethanol has been added before use, add Buffer PW along the wall of the tube to help flush residual salt from the wall), and centrifuge at room temperature at 12,000 rpm (~13,400×g) for 1 min. Discard the filtrate and put the DNA Spin Column into the collection tube;

11. Repeat step 10;

12. Reposition the DNA Spin Column in the Collection tube, centrifuge at room temperature at 12,000 rpm (~13,400×g) for 2 min, take out the DNA Spin Column and place it on a new 1.5 mL centrifuge tube;

13. Open the lid of DNA Spin Column and place it at room temperature for 3-5 min to make the residual ethanol volatilize completely;

14. Add 50-100 μL Buffer TE or Nuclease-free Water to the center of the membrane and place it at room temperature for 2 min. Centrifuge at room temperature at 12,000 rpm for 2 min and collect DNA solution. If you need higher concentration of DNA, the obtained solution can be re-added to DNA Spin Column, place it at room temperature for 2 min and centrifuge at room temperature at 12,000 rpm for 2 min, eluting DNA again.

1. Before operation, be sure to read this product manual carefully.

2. Wear a lab coat and disposable gloves during operation.

3. If the stool sample is dry, please increase the amount of Buffer SLA to 1 mL, or reduce the sample weight, upside down a few times and stand at room temperature for 3-5 min after adding the Buffer SLA, otherwise the DNA yield will be affected.

4. If need 95℃ incubation, please open the centrifuge tube lid once when temperature reaches 95℃, discharge the gas in the centrifuge tube, and then close the centrifuge tube lid tightly to prevent the gas in centrifuge tube expanding and bursting the lid, resulting in splashing of stool samples.

5. Some reagents are volatile or oxidized, long exposure to air should be avoided, the reagent should be closed in time after use.

For Research Use Only!