Stool or Soil Genomic DNA Extraction Kit

1. Components of the reagent kit

2. Storage

This reagent kit should be stored at room temperature (15-25℃) and in dry conditions, with a shelf life of 12 months. The DNA extraction purification columns can be stored for 1 year in a cool and dry environment. Lysozyme, Proteinase K, and RNase A contain preservatives, allowing transportation at room temperature, but for long-term storage, they should be kept at -20℃.

3. Instructions for Using the Reagent Kit

3.1 This reagent kit is intended for molecular biology research and should not be used for disease diagnosis or treatment.

3.2 Some components in the reagent kit contain irritants. Protective measures such as wearing protective clothing and goggles are recommended.

3.3 During the usage of this reagent kit, a high-speed centrifuge, water bath (metal bath), vortex mixer, anhydrous ethanol, sterile deionized water, and EP tubes need to be prepared by the user.

4. Introduction to the Reagent Kit

This kit provides a fast and efficient method for purifying microbial genomic DNA from soil, feces, and vomit samples. It is widely used for microbial DNA extraction from soil, feces, and vomit.

This kit effectively removes sample impurities and inhibitors, reducing inhibitory reactions in downstream experiments such as PCR. The entire purification process does not require toxic reagents such as phenol-chloroform. The extracted DNA can be directly used for PCR, Southern blotting, and other applications.

5. Experimental Principles and Procedures

6. Extraction Process

Before Starting the Experiment:

A. Reagent Buffer IV may precipitate under low-temperature conditions. We recommend heating at 65℃for 5 minutes. After the precipitate dissolves, it can be used normally.

B. Before use, add the specified amount of anhydrous ethanol to Wash Buffer 1 as indicated on the bottle label. Mark a check on the label to indicate the addition of anhydrous ethanol.

C. Elution Buffer is a 0.1x TE solution containing minimal amounts of EDTA. If EDTA might affect subsequent experiments, it is advisable to substitute Elution Buffer with sterile deionized water.

1. Sample Processing (Inhibitor Removal):

A. Fecal and Vomit Samples: Add approximately 200mg of the sample to be extracted, add 1ml of Humic Acid Removal Reagent I, vortex thoroughly for 1-2 minutes, centrifuge at 12,000 rpm for 2 minutes, discard the supernatant, add 560μl of Buffer IV to the pellet, invert and mix thoroughly, digest at 85℃ for 5 minutes, invert and mix 6-7 times during digestion, then proceed to step 2.

B. Soil Samples: Weigh approximately 0.1g-0.5g of sieved soil, add 500μl of Buffer IV and 100μl of Humic Acid Removal Reagent I, invert and mix thoroughly, digest at 85℃ for 5 minutes, invert and mix 6-7 times during digestion, then proceed to step 2.

(Note: Humic Acid Removal Reagent I/Humic Acid Removal Reagent II is mainly used for soils with high humic acid content, such as forest soils and sedimentary soils. Add 1ml of Humic Acid Removal Reagent I to the sample, vortex, shake for 1-2 minutes, centrifuge at 8,000 rpm for 2 minutes, discard the supernatant, then add 1ml of Humic Acid Removal Reagent II, vortex, centrifuge at 8,000 rpm for 2 minutes, discard the supernatant. This step can further reduce soil humic acid inhibitors but significantly reduce DNA yield.)

2. Centrifuge at 12,000 rpm for 2 minutes, transfer the supernatant to a new centrifuge tube (approximately 500μl liquid transferred), add 20μl Proteinase K (10 mg/ml), 20μl Lysozyme, and 20μl RNase A, and incubate at 65℃ for 2 minutes.
3. Add an equal volume of Reagent Buffer C plus (approximately 560μl liquid transferred) to the lysate, mix well. If a white precipitate appears, let it settle; it won’t affect subsequent experiments after the precipitate disappears.
4. Add an equal volume of ethanol to Reagent Buffer C plus (e.g., add 560μl of Reagent Buffer C plus, then add 560μl of ethanol), mix well. There may be precipitation, but it won’t affect subsequent experiments.
5. Add the obtained liquid to the DNA extraction purification column (sleeve) (approximately 650-700μl each time), centrifuge at over 8,000 rpm for 1 minute, discard the collected waste liquid, and reinsert the collection tube into the DNA extraction purification column (sleeve) for the next step.
6. Add 400μl Inhibitor Removal Buffer, centrifuge at over 8,000 rpm for 1 minute, discard the waste liquid, and reinsert the DNA purification column into the sleeve for the next step.
7. Add 500μl Wash Buffer 1 to the DNA extraction purification column (sleeve), centrifuge at 14,000 rpm (20,000×g) for 1 minute.
8. Add 300μl Wash Buffer 1 again to the DNA extraction purification column (sleeve), centrifuge at 14,000 rpm (20,000×g) for 2 minutes.
9. Place the DNA extraction purification column (sleeve) into a new centrifuge tube, open the lid, and incubate at 65℃ for 2 minutes. This step can be extended if necessary to evaporate ethanol as much as possible to prevent ethanol residue from affecting downstream experiments.
10. Drip 100μl Elution Buffer onto the membrane, centrifuge at 12,000 rpm for 2 minutes.

(Note: 1. Eluting DNA with 50μl Elution Buffer can increase DNA concentration but reduce total DNA yield; 2. The eluted DNA can be reapplied to the DNA extraction purification column, centrifuged again at 12,000 rpm for 2 minutes, and collected to increase DNA yield.)