Fast extraction method of sugarcane Ustilago scitaminea DNA
A technology of sugarcane smut and an extraction method, which is applied in the field of rapid extraction of sugarcane smut DNA, can solve problems such as economic losses, and achieve the effects of shortening cycle, reducing procedures and easy operation.
Inactive Publication Date: 2011-01-05
SUGARCANE RES INST OF YUNNAN ACADEMY OF AGRI SCI
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The disease was prevalent in some sugarcane areas and caused significant economic losses, and it is still causing economic losses to varying degrees
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[0010] The sugarcane smut diseased plants were collected from the field, and the smut spores on the diseased plants were directly extracted for DNA extraction. Proceed as follows:
[0011] 1. Take 0.5g of germ spores in a mortar, add 600μL of urea extraction mixture for grinding, the urea extraction mixture is composed of 7mol / L Urea, 50mmol / L Tris-HCl pH 8.0, 62.5mmol / L NaCl, 1% SDS ;
[0012] 2. Transfer the bacterial solution to a 2mL centrifuge tube, add 800μL of urea extraction mixture, fully shake and mix on a shaker, and centrifuge at 12000r / min for 5min;
[0013] 3. Take the supernatant into a new 2mL centrifuge tube and centrifuge at 12000r / min for 5min;
[0014] 4. Transfer the supernatant to another new 2mL centrifuge tube, add an equal volume of chloroform / isoamyl alcohol (24:1) solution, shake vigorously for several times to mix, and centrifuge at 12000r / min for 5min;
[0015] 5. Take the supernatant to a new 2mL centrifuge tube, add 2 volumes of isopropanol an...
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The invention relates to a fast extraction method of sugarcane Ustilago scitaminea DNA. The method comprises the following steps: taking 0.5g of the conidia of Ustilago scitaminea on the sugarcane with the Ustilago scitaminea, grinding the conidia of Ustilago scitaminea with a urea mixed extracting solution, shaking to mix evenly, centrifuging to take the supernatant, adding the same volume of chloroform / isopentyl alcohol solution, shaking to mix evenly, centrifuging to take the supernatant, adding isopropanol of which volume is twice of that of supernatant and NaAc of which volume is one tenth of that of the supernatant, standing at -20 DEG C for 1h, centrifuging to discard the supernatant, washing the precipitate with 70% ethanol solution, drying at the room temperature; dissolving the dried precipitate in 800mu L of double distilled water, adding 2mu L of RnaseA, heating with 37 DEG C of water bath for 1h, adding the same volume of chloroform / isopentyl alcohol solution, shaking to mix evenly, centrifuging to take the supernatant, adding isopropanol of which volume is twice of that of supernatant and NaAc of which volume is one tenth of that of the supernatant, standing at -20 DEG C for 1h, centrifuging to discard the supernatant, washing the precipitate with 70% ethanol solution, centrifuging to discard the supernatant, and drying at the room temperature to obtain the sugarcane Ustilago scitaminea DNA. Through the detection of agarose gel electrophoresis, the DNA bands are complete and bright.
Description
Technical field: [0001] The invention belongs to the field of plant protection, in particular to a rapid extraction method for sugarcane smut DNA. Background technique: [0002] Sugarcane smut (Ustilago scitaminea Syd.) has become one of the major diseases in sugarcane growing areas in the world. The disease was prevalent in some sugarcane areas and caused significant economic losses, and it is still causing economic losses to varying degrees. The severity of smut is closely related to the disease resistance and susceptibility of sugarcane varieties. The incidence rate of varieties with strong disease resistance is less than 10%, and the incidence rate of susceptible varieties can be as high as more than 50%. Yield losses are as high as 70%, and even as high as 80-90% in severe cases. [0003] Sugarcane smut is a fungal sugarcane disease caused by smut. Currently, various methods such as isolation and culture technology, serum (ELISA) or PCR technology are used to detect t...
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Inventor 卢文洁李文凤黄应昆李如丹王明强罗志明王晓燕
Owner SUGARCANE RES INST OF YUNNAN ACADEMY OF AGRI SCI