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A technology of grape canker bacteria and mycelium, applied in the field of molecular biology, can solve the problems of poor extraction quality, cost of money and energy, etc., and achieve high yield and high purity
Inactive Publication Date: 2019-03-05
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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Problems solved by technology
[0005] Using existing technology to extract RNA, polysaccharides and other impurities from grape canker mycelia, the extraction quality is not good, and cannot be directly used in downstream experiments. It needs to go through a large number of purification processes, which consumes money and energy.
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Embodiment 1
[0024] Embodiment 1, the method for extracting grape canker canker mycelia RNA
[0025] Consumables: Metal keys, mortars and pestles were soaked in 0.1N NaOH for 1 hour, washed with distilled water, and sterilized at 160°C for 6 hours; RNase-free sterilized tips and 1.5 mL centrifuge tubes (Axygen).
[0026] Reagents: Trizol (Qiagen); Chloroform; Isopropanol; 75% ethanol (prepared with DEPC treated water); Precipitation buffer (containing 1.2mol / L NaCl and 0.8mol / L disodium citrate 15H 2 O solution); RNase-free water;
[0027] Strain: wild-type P. vine canker strain CSS-01s, preserved in our laboratory (Liu Aifen, Zhang Xin, Zhang Wei, et al. Establishment of a rapid pathogenicity evaluation system for P. vine canker transformants[J]. Plant Protection, 2013, 2 :021).
[0028] 1) Strain preparation: Inoculate the preserved purified strain on a solid PDA medium plate with a sterile inoculation loop or toothpick, and culture overnight at 28°C for later use;
[0029] 2) Mycelia...
Embodiment 2
[0047] Consumables: Metal keys, mortars and pestles were soaked in 0.1N NaOH for 1 hour, washed with distilled water, and sterilized at 160°C for 6 hours; RNase-free sterilized tips and 1.5 mL centrifuge tubes (Axygen).
[0048] Reagents: Trizol (Qiagen); Chloroform; Isopropanol; 75% ethanol (prepared with DEPC treated water); Precipitation buffer (containing 1.2mol / L NaCl and 0.8mol / L disodium citrate 15H 2 O solution); RNase-free water;
[0049] Strain: wild-type P. vine canker strain CSS-01s, preserved in our laboratory (Liu Aifen, Zhang Xin, Zhang Wei, et al. Establishment of a rapid pathogenicity evaluation system for P. vine canker transformants[J]. Plant Protection, 2013, 2 :021).
[0050] 1) Strain preparation: Inoculate the preserved purified strain on a solid PDA medium plate with a sterile inoculation loop or toothpick, and culture overnight at 28°C for later use;
[0051] 2) Mycelia suspension preparation: after the mycelium obtained in step 1) was eluted with PD...
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Abstract
The invention discloses a method for extracting Botryosphaeria dothidea hypha RNA (ribonucleic acid). The method comprises the following steps: grinding Botryosphaeria dothidea hyphae preserved in liquid nitrogen into powder in the liquid nitrogen; transferring the powder into a precooled centrifuge tube, adding 1mL of Trizol leaching liquor to every 90-110mg of powder, uniformly mixing by shaking, and standing at room temperature; adding chloroform, oscillating intensely, standing at room temperature, and centrifugating; putting the supernate into a new centrifuge tube, adding isopropanol and a precipitate buffer solution, and uniformly mixing in a reversed mode; centrifugating; discarding the supernate, adding 1mL of a 75 vol% ethanol solution to clean the RNA precipitate block, centrifugating, and discarding the supernate; repeating the cleaning operation; and discarding the ethanol, opening the cap, standing at room temperature for 10-15 minutes or carrying out vacuum drying to completely volatilize the ethanol, thereby obtaining the precipitate RNA product. The RNA extracted by the method has the advantages of high purity and high yield.
Description
technical field [0001] The invention belongs to the field of molecular biology, and mainly relates to a method for efficiently extracting fungal total RNA by applying an improved Trizol method. Background technique [0002] Grape (Vitis vinifera L.) has always been one of the most important fruits in the world, accounting for more than 10% of the global fruit planting area. It can not only be eaten fresh but also be processed for wine making, drying and juice making. By the end of 2013, my country's grape cultivation area has reached 714,600 hm 2 , with a grape production of 11.55 million tons, ranking first in the world's grape production since 2010. [0003] Grape disease is one of the most important factors affecting grape yield and quality. According to statistics, there are more than 40 kinds of diseases that endanger grape production in my country. According to their harmful parts, grape diseases can be divided into grape leaf diseases, fruit diseases, branch diseases...
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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12R1/645
CPCC12Q1/6806C12Q2523/32
Inventor 李兴红邢启凯燕继晔张玮
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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