Method for extracting total DNAs of soil microorganisms at high purity
A soil microbial, high-purity technique for applications in the field of molecular ecology
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Embodiment 1
[0051] PVP pretreatment-CTAB-lysozyme-protease-SDS-repeated freeze-thaw method. Take 5g of soil sample and add 30mL of 20g / L sodium metaphosphate buffer (containing 10g / LPVP-K30; pH 8.5), oscillate on a shaking table for 15min, centrifuge at 8500r / min for 5min, take the precipitate, and repeat washing 3 times; take the precipitate, add 13.5 mL of DNA extraction solution I and a little quartz sand, vortex for 3 minutes; add 150 μL of 100 mg / mL lysozyme, mix well, and incubate in a shaker at 37 ° C and 230 r / min for 1 hour; then add 15 μL of 20 mg / mL proteinase K, mix well, Add 1.5mL of 10% SDS in water bath at 37°C for 1h; add 1.5mL 10% SDS, bathe in water at 65°C for 1h; freeze-thaw three times, centrifuge at 8500r / min for 10min; take the supernatant, add an equal volume of phenol / chloroform / isoamyl alcohol (volume ratio 25: 24:1) extraction, 8500r / min centrifugation for 10min; take the supernatant, add an equal volume of chloroform / isoamyl alcohol (volume ratio 24:1) to extra...
Embodiment 2
[0053] PVP pretreatment-CTAB, CaCl2, BSA-lysozyme-protease-SDS-repeated freezing and thawing method Take 5g soil sample and add 20g / L sodium metaphosphate buffer (containing 10g / LPVP-K30; pH 8.5) 30mL, shake the table 15min, centrifuge at 8500r / min for 5min, take the precipitate, repeat washing 3 times; take the precipitate, add 13.5mL DNA extraction solution II and a little quartz sand, vortex shaker for 3min; add 100mg / mL lysozyme 150μL, mix well, 37℃ , Incubate in a shaker at 230r / min for 1h; then add 15μL of 20mg / mL proteinase K, mix well, and bathe in water at 37°C for 1h; add 1.5mL 10% SDS, bathe in water at 65°C for 1h; repeat freeze-thawing 3 times, and centrifuge at 8500r / min for 10min; Get the supernatant, add an equal volume of phenol / chloroform / isoamyl alcohol (volume ratio 25:24:1) for extraction, and centrifuge at 8500r / min for 10min; get the supernatant, add an equal volume of chloroform / isoamyl alcohol (volume Ratio 24:1) and extract again, centrifuge at 8500r / ...
Embodiment 3
[0055]CTAB, PVP, CaCl2, BSA-lysozyme, protease-SDS-repeated freeze-thaw method. Take 5g soil sample, add 13.5mL DNA extraction solution III and a little quartz sand, vortex shaker for 3min; add 150μL 100mg / mL lysozyme, 15μL 20mg / mL proteinase K, mix well, incubate for 1h at 37℃, 230r / min shaker Add 15 μL of 20 mg / mL proteinase K, mix well, and bathe in water at 37°C for 1 hour; add 1.5 mL of 10% SDS, bathe in water at 65°C for 1 hour; Extract with phenol / chloroform / isoamyl alcohol (volume ratio 25:24:1), centrifuge at 8500r / min for 10 min; take the supernatant, add an equal volume of chloroform / isoamyl alcohol (volume ratio 24:1) to extract again, Centrifuge at 8500r / min for 10min, take the supernatant, add 0.7 times the volume of isopropanol and 0.1 times the volume of NaAc to the supernatant, place it overnight at -20°C, centrifuge at 8500r / min, 4°C for 20min, wash the precipitate with 70% ethanol, Centrifuge at 12500r / min for 10min, dry the precipitate at room temperature,...
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