| Product Description | Product content：
| Component |
S665549
50 preps |
| Buffer SW |
60 ml |
| Buffer SL |
60 ml |
| Buffer GL |
50 ml |
| Buffer GW1（concentrate） |
2X13 ml |
| Buffer GW2（concentrate） |
15 ml |
| Buffer GE |
15 ml |
| Spin Columns DM |
50 |
| with Collection Tubes |
50 | |
Product Introduction
This kit is suitable for extracting total DNA from fecal samples, including the total DNA of cells, bacteria, parasites, and viruses in the samples, as well as samples containing high concentrations of PCR reaction inhibitors. This product can process up to 300 mg of fecal samples and purify to obtain mainly 20-30 kb DNA fragments. The purification process does not require toxic solvents such as phenol or chloroform, and does not require ethanol precipitation. High purity DNA can be obtained within one hour. This reagent kit adopts a unique buffering system to efficiently bind DNA from the lysis solution to the adsorption column. At the same time, protein impurities and other organic compounds that inhibit downstream reactions in feces can flow through the membrane. Inhibitors of PCR and enzyme reactions, as well as residual impurities, can be effectively removed through two washing steps. Finally, high-purity DNA can be obtained by washing with low salt buffer or water. The purified DNA can be directly used for downstream experiments such as enzyme digestion, PCR, Real Time PCR, library construction, Southern Blot, and molecular labeling. Preparation and important precautions before the experiment
1. Samples should avoid repeated freeze-thaw cycles, otherwise it may result in smaller extracted DNA fragments and a decrease in extraction volume.
2.Before the first use, anhydrous ethanol should be added to Buffer GW1 and GW2 according to the instructions on the reagent bottle label.
3. Before use, please check whether there is crystallization or precipitation in Buffer SL and Buffer GL. If there is crystallization or precipitation, please dissolve Buffer SL and Buffer GL again in a 56 ℃ water bath.
4. If downstream experiments are sensitive to RNA contamination, 4 can be added after adding Buffer SL μ RNase A of DNase Free (100 mg/ml) is not provided in this kit. If needed, it can be ordered separately from our company, item number: S665549 Operation steps
1. Take a fecal sample of 100-300 mg and place it in a centrifuge tube (provided by oneself).
2. Add 1 ml of Buffer SW and vortex for 3-5 minutes to evenly disperse the sample in the solution. Centrifuge at 12000 rpm (~13400 × g) for 1 minute and discard the supernatant.
3. Add 1 ml of Buffer SL and vortex for 3-5 minutes to evenly disperse the sample in the solution. Take a water bath at 65 ℃ for 20 minutes and vortex for 15 seconds every 5 minutes. Note: To remove RNA, add 4 after completing the above steps μ RNase A solution (product number: CW0601S) with a concentration of 100 mg/ml, shake well and let stand at room temperature for 5-10 minutes.
4.Centrifuge at 2000 rpm for 3 minutes and transfer the supernatant to a new centrifuge tube (provided by oneself).
5. Add an equal volume of Buffer GL to the supernatant, invert and mix 15-25 times, and leave on ice for 5 minutes. Centrifuge at 12000 rpm for 5 minutes. Attention: At this time, the liquid may be in a transparent or turbid state, which does not affect the experiment. 6. Add the supernatant obtained in step 5 to the spin columns DM that have been loaded into the collection tube. If the solution cannot be added at once, it can be transferred multiple times. Centrifuge at 12000 rpm (~13400 × g) for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.
7. Add 500 to the adsorption column μ Buffer GW1 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. 8. Repeat step 7.
9. Add 500 to the adsorption column μ Buffer GW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. 10.12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly air dry. Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).
11. Place the adsorption column in a new centrifuge tube (self provided) and add 50-100 drops of suspended droplets to the middle of the adsorption column μ L Buffer GE or sterilized water, leave at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, collect DNA solution, and store DNA at -20 ℃.
Note: 1) If downstream experiments are sensitive to pH or EDTA, they can be washed off with sterilized water. The pH value of the eluent has a significant impact on the elution efficiency. If water is used as the eluent, its pH value should be ensured to be between 7.0-8.5 (NaOH can be used to adjust the pH value of the water to this range). When the pH value is below 7.0, the elution efficiency will be reduced 2) Incubating at room temperature for 5 minutes before centrifugation can increase yield. 3) Use an additional 50-100 μ Further washing with buffer GE or sterilized water can increase yield.
4) If you want to increase the final concentration of DNA, you can add the DNA eluent obtained in step 11 back onto the adsorption membrane and repeat step 11; It is possible to increase the final concentration of DNA, but it may reduce the total yield. If the amount of DNA is less than 1 μ g. Recommended 50 μ Wash with buffer GE or sterilized water.
5) DNA stored in water can be affected by acidic hydrolysis. If long-term storage is required, it is recommended to elute with Buffer GE and store at -20 ℃.
6) The residual trace PCR inhibitors in the genomic DNA template may have adverse effects on the PCR reaction, which can be solved by diluting DNA by 2-10 times.
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