Stool. Plant. Microbe. Extract Fecal DNA for Microbiome Analysis.

Stool is an accessible and noninvasive source of biospecimen. Fecal DNA/RNA originated from the host, intestinal bacteria, viruses, fungi, or parasites, and incompletely digested foods are molecular fingerprints of the host and its health. AquaStool™ is a multifunctional aqueous reagent for fecal sample stabilization, DNA and RNA extraction and PCR inhibitor removal. It may be used to extract fecal DNA for non-invasive genotyping of transgenic animals. AquaStool may be used to preserve and extract DNA/RNA from human stools for host and gut microbiome research. AquaStool may also be used to extract DNA from tree tissues, which are particularly challenging with existing DNA extraction methods

AquaStool Brief Protocol

(Download AquaStool User Manual for protocols to extract DNA/RNA from mouse fecal pellets, human stools, wood tissues, and more)

1. Extract the DNA/RNA

Add 50 mg of human fecal sample to a 1.5-ml microfuge tube preloaded with 0.5 ml AquaStool solution and ~100 ug of white sand. Vortex the tube upside down at top speed for a few minutes (use a multichannel bead beater, if available). Centrifuge at 14,000 xg for 5 min to pellet the fecal debris. Optional: Add 250 ul of AquaRemove (#1208, order separately and dilute it with an equal volume of isopropanol before use) to the clear lysate. Vortex to mix well and centrifuge at 14,000 xg again for 5 min to pellet the fecal debris.

2. Pellet the DNA/RNA

Transfer 0.6 ml cleared lysate to a 1.5-ml tube and add 0.8 volume (0.42 ml) isopropanol. Vortex and centrifuge at 14,000 xg for 5 min to pellet the DNA/RNA. Decant to discard the supernatant. Gently fill up the tube and its lid with 70% ethanol from a squirt bottle and then decant to discard the ethanol solution. Repeat the 70% ethanol rinse once. Flip the tube forcefully a few times and tap it on a paper towel to remove residual ethanol. Let the DNA/RNA pellet air-dry for 5-10 min.

3. Solubilize the DNA/RNA

Add 0.4 ml deionized water to the DNA/RNA pellet, pipet and vortex to fully disperse the pellet. Incubate at 22 °C for 15 min and then centrifuge at 14,000 xg for 5 min to pellet any insoluble. Transfer the clear DNA/RNA solution to a new tube and store at it –20 or -80 °C (Note: Centrifuge the frozen DNA solution to remove any insoluble before its use in a PCR reaction).

FAQ about AquaStool

1. How should I store the AquaStool solution?

AquaStool may be stored at 22 °C for 12 months. If AquaStool becomes precipitated when exposed to low temperature, you may incubate it at 37-50 °C for 15-20 min to resolubilize the reagent.

2. Why shouldn’t I use Bleach to disinfect AquaStool preserved fecal specimen?

AquaStool contains guanidine thiocyanate. It may react with Bleach (sodium hypochlorite) and release toxic gases.

3. How should I air-dry the mouse fecal samples?

Air-dried mouse fecal samples can be stored long term at room temperature for future genotype verification. To air-dry a mouse fecal pellet, incubate the fecal pellet in an opened microfuge tube on a dry heat bloc at 37 °C for 24 hours.

4. Why is my DNA/RNA solution showing a strong absorption below A260?

It is likely due to trace amount of guanidine salt contamination. If it interferes with your downstream applications, you may further purify the extracted DNA/RNA with a silica spin column (e.g., a plasmid miniprep column). Simply add an equal volume of 4 M GuHCl and 1M NaOAc (pH unadjusted, ~7.0) to your DNA/RNA solution (may contain the insoluble pellet) and load it into the spin column, centrifuge to allow DNA/RNA binding to the silica membrane, wash the column with 0.6 ml 75% EtOH, and elute the DNA/RNA in 50 μl of deionized water or TE buffer.

5. My mouse transgene was not amplified well, how may I improve it?

You may try the following to improve mouse fecal DNA amplification: (a) after freezing the fecal DNA solution at -20 °C, re-centrifuge it to pellet and remove any insoluble, which may contain PCR inhibitors; (b) reduce the amount of fecal DNA used per PCR reaction (i.e., try using 0.5, 0.25, 0.1 and 0.01 ml of extracted fecal DNA per PCR reaction); (c) increase PCR cycles to 65; (d) add 1 mM DTT and 0.1 mg/ml BSA to the PCR reaction; (e) use a gel imager to visualize faint amplicon bands; (f) use AquaRemove (#1208, order separately) with AquaStool to purify fecal DNA (see “Human fecal DNA/RNA extraction” for details); and (g) further purify the fecal DNA with a silica spin column as outlined in #4 Question and Answer above.

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