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uitable for extracting total DNA from feces, soil, water samples, biofilms, swab washes, biological body fluids The total DNA of various microorganisms in the lysed sample can be bound to the Spin Column, the degraded protein and PCR inhibitors such as humic acid are filtered out, and the genomic DNA is washed by Buffer WB and eluted with Buffer TE for various molecular biology experiments. Environmental DNA Isolation /Extraction Kit Instructions
Composition
Environmental DNA Extraction Kit Cat. No. | 50 Preps 4107050 |
2 ml Sample Tubes Spin Columns Proteinase K Buffer PD Buffer ST Buffer TE Buffer P Buffer WB (concentrate) Instructions | 50 50 1.2 ml 55 ml 12 ml 20 ml 28 ml 10 ml 1 |
Storage
1. Proteinase K can be transported and stored at room temperature (0~30℃) with a validity period of 1 year, if stored at -20℃ the validity period can be extended to 2 years.
2. If other reagents and items are stored at room temperature (0~30℃), they can keep the performance unchanged for 2 years, and if stored at 2~8℃, the validity period can be extended to more than 2 years (the product stored at 2~8℃ should be restored to room temperature before use).
Introduction
This product is suitable for extracting total DNA from feces, soil, water samples, biofilms, swab washes, biological body fluids. The total DNA of various microorganisms in the lysed sample can be bound to the Spin Column, the degraded protein and PCR inhibitors such as humic acid are filtered out, and the genomic DNA is washed by Buffer WB and eluted with Buffer TE for various molecular biology experiments.
Equipment And Reagents to Be Supplied by Users
1． Absolute ethanol, isopropanol and deionized purified water may be required.
2． 1.5 ml and 2 ml centrifuge tubes.
3． Pipettes and tips (to avoid contamination between samples, use tips with filters).
4． Disposable gloves and protective equipment and tissues.
5． Microcentrifuge (s) (with rotor for 1.5 ml and 2 ml tubes).
6． Sample homogenizer, water bath, and vortexer.
Preparation Before Use
1． If the centrifuge has refrigeration function, set the temperature to 25℃.
2． Set the water bath temperature to 70℃ and incubate Buffer PD, Buffer ST, and Buffer TE at 70℃.
3． Add absolute ethanol to Buffer WB according to the instructions on the label of the reagent bottle and tick the box on the label to mark "Ethanol Added".
Protocol
1. Samples were taken according to the sample type in the chart and added to a 2 ml sample tube. Add 1 ml Buffer PD, tighten the cap, and place in a sample homogenizer for 30 sec at full speed, or vortex vigorously for 3 min if no sample homogenizer is available.
Sample type | Maximum amount of input |
soil | 500 mg |
feces | 200 mg |
Liquid samples (water samples, biological fluids, swab wash fluids, etc., or bacterial or cell samples suspended in nucleic acid protective agents). | 500 μl |
* If lyophilized soil powder is used, an additional 100 μl deionized purified water should be added to facilitate the suspension of soil particles.
2. Add 20 μl Proteinase K, cover the lid and mix well, incubate at 70℃for 15 min. During the incubation, vortex vigorously every 5 min for 30 sec.
3. Add 200 μl Buffer ST, vortex for 30 sec, centrifuge at 13,000 rpm for 10 min.
4. Transfer the supernatant (about 1 ml or so) into a clean 2 ml centrifuge tube (not provided).
5. Add 800 μl isopropanol, gently invert 4~6 times and mix well. Centrifuge at 13,000 rpm for 10 min.
6. Discard the supernatant and spin down the residual supernatant to the bottom of the centrifuge tube. discard the residual supernatant with a tip and retain the pellet at the bottom of the tube.
7. Add 100 μl 70℃-incubated Buffer TE and vortex until the pellet is completely dissolved.
8. Add 500 μl Buffer P and gently invert 4~6 times to mix well. Transfer the mixture to a Spin Column, close the lid, and centrifuge at 12,000 rpm for 30 sec.
9. Discard the filtrate, place the Spin Column back into the 2 ml collection tube, add 600 μl Buffer WB to the Spin Column, close the lid, and centrifuge at 12,000 rpm for 30 sec.
* The filtrate does not need to be completely discarded, if you want to avoid contamination of the centrifuge by the filtrate adhered to the mouth of the collection tube, you can slap the 2 ml collection tube upside down on a paper towel once.
* Ensure that absolute ethanol has been added to Buffer WB.
10. Discard the filtrate, place the Spin Column back into the 2 ml collection tube, and centrifuge at 14,000 rpm for 1 min.
* If the centrifuge does not reach 14,000 rpm, centrifuge at full speed for 2 min.
* Do not omit this step, otherwise the subsequent PCR effect may be affected due to the ethanol mixed in the purified DNA.
11. Discard the 2 ml collection tube, place the Spin Column in a clean 1.5 ml centrifuge tube, add 100~200 μl 70℃ incubated Buffer TE in the Spin Column center, close the lid, incubate at room temperature for 1 min, and centrifuge at 12000 rpm for 30 sec.
* If the centrifuge does not have a leak-proof lid, change the centrifugation condition to 8000 rpm for 1 min to avoid damage to the centrifuge due to the detachment of the cap of the 1.5 ml centrifuge tube.
12. Discard the Spin Column, the eluted DNA can be immediately used in various molecular biology experiments or stored at -20℃ for later use.
* When used for PCR amplification, the amount of DNA used as template should not exceed 1/10 of the final reaction volume (e.g., if the final reaction volume is 50 μl, the amount of DNA should not exceed 5 μl).
