Extraction method of guava phyllospheric microganism genome DNA (Deoxyribonucleic Acid)
A guava leaf and extraction method technology, applied in the field of bioengineering, can solve the problems of backward plant phyllosphere microorganisms and little understanding of the characteristics of non-pathogenic phyllosphere microorganisms, and achieve high extraction rate, broad market prospects, and simple operation steps Effect
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Embodiment 1
[0032] A method for extracting guava phyllosphere microbial genome DNA, comprising the following steps:
[0033] (1) Add 2 g of guava leaves to 4 mL of phosphate buffer solution, add 0.3 g of quartz sand and 0.2 g of 1mm glass beads, shake on an adjustable vortex mixer for 20 min, and then perform ultrasonic (20 min, 300 W) treatment after high-intensity vortex shaking, and centrifuge Obtain the mixed liquor containing phyllosphere microorganisms after removing guava leaves;
[0034](2) Add 150uL TENP solution to the mixture containing phyllosphere microorganisms, -20°C, 18min, 45°C for 5min, freeze-thaw 3 times, then add 5uL 100mg / mL lysozyme and proteinase K, at 37°C Water bath for 40 minutes, invert the centrifuge tube up and down 4 times, then add 0.5mL SDS buffer, bath in 68°C water for 40 minutes, invert the centrifuge tube up and down 4 times, centrifuge at 15°C, 1000rpm for 10min to obtain precipitate a and supernatant a; a replace the mixed solution containing phyllo...
Embodiment 2
[0040] A method for extracting guava phyllosphere microbial genome DNA, comprising the following steps:
[0041] (1) is identical with embodiment 1 step (1);
[0042] (2) Add 150uL TENP solution to the mixture containing phyllosphere microorganisms, -20°C, 18min, 45°C for 5min, freeze-thaw 3 times, then add 5uL 100mg / mL lysozyme and proteinase K, at 37°C Water bath for 40 minutes, invert the centrifuge tube up and down 4 times, then add 0.5mL SDS buffer, bath in 68°C water for 40 minutes, invert the centrifuge tube up and down 4 times, centrifuge at 15°C, 1000rpm for 10min to obtain precipitate a and supernatant a; a replace the mixed solution containing phyllosphere microorganisms and repeat the above steps twice; wherein, the composition of the TENP solution includes: 50mM Tris, 20mM EDTA, 100mM NaCl, 0.01g / mLPVP, the pH of the TENP solution is 10; the composition of the SDS buffer Including: 100mmoL / LTris, 100mmoL / L EDTA-2Na, 200mmoL / L NaCl, volume fraction of 2% PVP, volu...
Embodiment 3
[0048] A method for extracting guava phyllosphere microbial genome DNA, comprising the following steps:
[0049] (1) is identical with embodiment 1 step (1);
[0050] (2) Add 150uL TENP solution to the mixture containing phyllosphere microorganisms, freeze and thaw repeatedly 4 times at -20°C for 18min and 45°C for 5min, then add 5uL of 100mg / mL lysozyme and proteinase K at 37°C Water bath for 40 minutes, invert the centrifuge tube up and down 4 times, then add 0.5mL SDS buffer, bath in 68°C water for 40 minutes, invert the centrifuge tube up and down 4 times, centrifuge at 15°C, 1000rpm for 10min to obtain precipitate a and supernatant a; a replace the mixed solution containing phyllosphere microorganisms and repeat the above steps once; wherein, the components of the TENP solution include: 50mM Tris, 20mM EDTA, 100mM NaCl, 0.01g / mLPVP, the pH of the TENP solution is 10; the components of the SDS buffer include : 100mmoL / LTris, 100mmoL / L EDTA-2Na, 200mmoL / L NaCl, the volume ...
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