Strong stool DNA extraction kit instruction manual

Strong stool DNA extraction kit

Introduction

The PowerFecalTM DNA Isolation kit is designed for easy and fast fecal, intestinal contents, biosolid sample microbes and host total DNA. Based on the PowerSoilTM DNA Isolation Kit, the Inhibitor Removal Technology® is used for both soil and fecal samples. IRT technology efficiently removes inhibitors such as polysaccharides, heme and bile salts commonly found in fecal samples. Pure final DNA can be used directly in all downstream experiments.

Operation preview

Fecal or biosolids are recommended to be loaded 0.25g. The sample was loaded into a separate 2 ml Bead Beating Tube containing garnet beads. The mechanical collision of the grinding beads and the cracking effect of the chemical reagent on the cell membrane promote the lysis of the host and the microbial cells, and the efficient extraction effect is sufficient to cope with the most difficult type of microorganisms. The patented Inhibitor Removal Technology® then removes the PCR inhibitors commonly found in stool samples. Total genomic DNA was adsorbed onto a silica gel filter. After rinsing and elution, the pure final DNA can be directly used for further applications such as PCR, qPCR, and second-generation sequencing.

Equipment requirements

Micro Centrifuge (13000g)

Pipette (60μl – 750μl)

Vortex-Genie®2 Vortex (MO BIO Item #13111-V-220) Vortex Adapter (MO BIO Item #13000-V1-24)

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The components are stored at room temperature (15-30 ° C).

Kit components

â–³ Please check the reagents one by one before the experiment !

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The components are stored at room temperature (15-30 ° C).

Steps

1. Add 0.25g of feces or biosolids to the Dry Bead Tube .

Note: Reduced loading (0.1g) of stool samples (eg, feces) and certain bird feces with higher moisture, polysaccharides, and protein levels can increase DNA yield and purity.

2. Add 750 μl Bead Solution to the Dry Bead Tube . Mix gently by vortexing.

What happened in this step: After adding the sample to the Bead Tube, the next step is sample homogenization and cell lysis. The garnet beads and the lysis lysis buffer dissolve and spread the sample.

3. Check the C1 solution before use . If there is precipitation, water bath at 60 ° C until all dissolved.

What happened in this step: The C1 solution contains SDS. A lower temperature will result in a white precipitate on the bottom of the bottle. The 60°C water bath re-dissolves SDS and does not impair the performance of SDS and other components. Can be used hot.

4. Add 60 μl of C1 solution, top upside down or gently vortex to mix.

What happens in this step: The C1 solution contains SDS and other lysis reagents to aid cell lysis. SDS is a detergent that degrades fatty acids and oils on cell membranes.

5. Heat in a 65 ° C water bath for 10 min.

What happened in this step: the stool sample mixed with many polysaccharides, lipids, salts and cells. Heating accelerates the reaction of the lysis buffer with these substances

At a rate that assists in lysing cells.

6. Place the Bead Tubes horizontally on the vortex adapter (MOBIO item number: 13000-V1-24). The maximum speed was continuously vortexed for 10 min.

What happens in this step: the grinding beads and microbial cells oscillate under the lysis reagent, which can rupture the cells. MOBIO's vortex adapter is a simple and effective accessory that snaps the Tube to the vortex during vortexing. You can also tape the Tube to the adapter, but it may loosen during the oscillation process, affecting the consistency of conditions between the samples and reducing the yield.

7. Centrifuge at 13000g for 1 min.

8. Transfer the supernatant to a clean 2ml Collection Tube (provided in the kit). Approximately 400-500 μl of supernatant can be obtained.

What happened in this step: about 400-500 μl of supernatant was obtained in this step. The expected volume is related to the initial amount of the sample and does not affect the extraction efficiency.

9. Add 250 μl of C2 solution and mix gently by vortexing. Incubate at 4 ° C for 5 min.

What happened in this step: The C2 solution is part of the patented Inhibitor Removal Technology® (IRT). It contains reagents that precipitate non-DNA organic and inorganic substances such as polysaccharides, cell debris and proteins. These organic and inorganic substances affect DNA purity and inhibit downstream experiments and must be removed.

10. Centrifuge at 13000g for 1 min.

11. Avoid sedimentation and transfer up to 600μl of supernatant to a clean 2ml Collection Tube (provided in the kit).

What happened in this step: precipitation of non-DNA organic and inorganic components such as polysaccharides, cell debris, and proteins. To achieve optimal DNA yield and quality, avoid sedimentation when transferring supernatant.

12. Add 200 μl of C3 solution and vortex gently. Incubate at 4 ° C for 5 min. 13. Centrifuge at 13000g for 1 min.

14. Avoid sedimentation and transfer the supernatant to a clean 2ml Collection Tube (provided in the kit). The transferred supernatant should not exceed 750 μl.

What happened in this step: precipitation of non-DNA organic and inorganic components such as polysaccharides, cell debris, and proteins. To achieve optimal DNA yield and quality, avoid sedimentation when transferring supernatant. The supernatant of the transfer should not exceed 750 μl, otherwise it will affect the adsorption binding of subsequent DNA.

15. Shake the C4 solution before use. Add 1200 μl of C4 solution to the supernatant and vortex for 5 s to mix.

What happened in this step: the C4 solution is a high concentration salt solution. The DNA can be selectively adsorbed onto the silica gel membrane. Non-DNA organic and inorganic substances are not adsorbed, but a small amount remains on the membrane.

16. Load 650 μl of supernatant into a Spin Filter and centrifuge at 13,000 g for 1 min. Discard the filtrate and repeat loading the supernatant.

Note: A total of 3 supernatants need to be loaded.

High-throughput options: Step 16 becomes very time consuming when processing large numbers of samples at once. Therefore MOBIO has developed a vacuum pump suction method. need

Additional purchase of vacuum pump adapter (MOBIO Item No.: 11992). The flat bottom Spin filter quickly completes the filtering action with the help of a vacuum pump adapter.

What happens in this step: DNA is selectively adsorbed onto the spin column silica membrane in a high concentration of salt. Impurities pass through the filter and the remaining DNA remains on the filter.

17. Add 500 μl of C5 solution and centrifuge at 13,000 g for 1 min.

What happens in this step: C5 is an alcohol-containing rinse buffer that can further rinse the DNA on the spin column silica filter. The rinse buffer also removes small amounts of impurities and other impurities remaining on the silica gel filter.

18. Discard the filtrate.

What happened in this step: the filtrate contained no DNA.

19, 13000g centrifuge again for 1min.

What happened in this step: centrifuge again to remove residual C5 solution (alcohol-containing rinse buffer). Alcohol-containing C5 solutions must be completely removed to avoid subsequent DNA applications such as PCR, digestion, and gel electrophoresis.

20. Carefully transfer the Spin Filter to a clean 2ml Collection Tube (provided in the kit). Avoid getting any C5 solution .

21. Add 100 μl of C6 solution to the center of the white column of the spin column. You can also elute DNA from a silica gel spin column using sterile DNA-Free PCR grade pure water or TE buffer.

Note: The best DNA yield is obtained with 100μl C6 solution. In order to concentrate the DNA, the C6 solution can be used in a minimum of 50 μl. A solution volume of less than 50 μl of C6 will seriously affect the elution efficiency.

What happened in this step: Add the C6 solution (sterilized elution buffer) to the center of the white filter of the spin column and wet the entire filter to get the best elution. The C6 solution (elution buffer) was passed through a filter, and the DNA was selectively adsorbed onto the filter in a high concentration of salt and eluted in a low salt environment of C6 solution (10 mM Tris).

22, centrifuge at 13000g for 1min, discard the Spin Filter.

The DNA in the tube can now be used directly in downstream experiments without further processing.

DNA is recommended to be stored frozen at -20 ° C ~ -80 ° C. The C6 solution does not contain EDTA. To concentrate DNA, refer to the following troubleshooting points.

Thank you for choosing PowerFecal TM DNA Isolation Kit!

Difficult to doubt

Sample throughput

This kit is designed to extract 0.25g of stool sample each time. We do not recommend loading more than 0.25g. For some stool samples containing large amounts of protein, lipids and polysaccharides, a small loading (0.1g) increases DNA yield and purity.

Fecal sample with a large water content

If the stool sample contains more water, add the sample to the Bead tube and centrifuge at 10,000g for 30s. Remove all free water. Then proceed to step 2.