A kind of extraction method of soil microbial genome
A soil microorganism and extraction method technology, applied in recombinant DNA technology, DNA preparation and other directions, can solve the problems of low DNA yield, unstable DNA performance, and many operation steps, and achieve high gene purity, fewer reagent types, and improved yield. Effect
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Embodiment 1
[0043] (1) Take 5g of soil sample in a 50mL centrifuge tube, add 2mol / L Ca(OH) 2 Aqueous solution 5mL, shake for 2min to disperse and mix the soil sample to obtain the soil mixture;
[0044] (2) Add 5mL of 0.2mol / L sodium phosphate solution and 5mL of 0.2mol / L NaOH solution to the soil mixture, vortex for 5min to fully release the cells in the soil, then centrifuge at 10000g for 5min to collect the precipitate .
[0045] (3) Put 0.5 g of the precipitate into a test tube, then add 500 μL of DNA extraction solution (10 mmol / L Tris-HCl, pH7.5, 10 mmol / L EDTA, 100 mmol / L NaCl, 5% SDS) into the test tube, and then add Add 30 μL of lysozyme solution (50 mg / mL) and 20 μL of proteinase k solution (20 mg / mL) in a water bath at 70°C for 30 min to break the cell wall and release the DNA to obtain solution A.
[0046] (4) Add a mixed solution of chloroform and isoamyl alcohol equal to the volume of solution A to solution A (the volume ratio of chloroform to isoamyl alcohol is 24:1), tur...
Embodiment 2
[0055] (1) Take 5g of soil sample in a 50mL centrifuge tube, add 2mol / L Ca(OH) 2 10mL aqueous solution, shake for 2min to disperse and mix the soil sample, and obtain the soil mixture;
[0056] (2) Add 5mL of 0.2mol / L sodium phosphate and 5mL of 0.2mol / L NaOH solution to the soil mixture, vortex for 5min to fully release the cells in the soil, and then centrifuge at 8000g for 5min to collect the precipitate.
[0057] (3) Add 0.5 g of the precipitate to the test tube, then add 750 μL of DNA extraction solution (10 mmol / LTris-HCl, pH7.5, 10 mmol / L EDTA, 100 mmol / L NaCl, 5% SDS) to the test tube, and then add 30 μL Lysozyme solution (50 mg / mL) and 20 μL of protease solution k (20 mg / mL), and then in a water bath at 70 ° C for 35 min to break the cell wall and release the DNA to obtain solution A.
[0058] (4) Add a mixture of chloroform and isoamyl alcohol equal to the volume of solution A to solution A (the volume ratio of chloroform to isoamyl alcohol in the mixture of chlorof...
Embodiment 3
[0068](1) Add the soil to the centrifuge tube, and add a calcium hydroxide aqueous solution with a concentration of 1mol / L, shake for 10 minutes to disperse the soil sample, and obtain a soil mixture; wherein, the ratio of calcium hydroxide in the soil to the calcium hydroxide aqueous solution For 5g: 20mmol;
[0069] (2) Add 0.1mol / L sodium phosphate solution and 0.2mol / L sodium hydroxide solution to the soil mixture, shake for 5 minutes to release the cells in the soil, and then centrifuge to collect the precipitate; wherein, the soil and phosphate solution The ratio of sodium hydroxide in phosphate and sodium hydroxide solution is 5g: 0.1mmol: 0.1mmol;
[0070] (3) Add DNA extract, lysozyme solution and proteinase k solution to the precipitate, then heat at 60°C for 1 h to obtain solution A; wherein, the ratio of the precipitate, DNA extract, lysozyme solution and proteinase k solution is 0.5 g: 1500 μL: 30 μL: 60 μL; the DNA extraction solution includes 10mmol / L Tris-HCl ...
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