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Method for extracting total microbial DNA from strong-flavor yeasts
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A strong-flavored, microbial technology, applied in the field of molecular biology, to achieve the effect of clear and bright bands, obvious removal effect and low cost
Inactive Publication Date: 2014-12-03
LUZHOU PINCHUANG TECH CO LTD
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Problems solved by technology
At present, there is no DNA extraction method suitable for Luzhou-flavor Daqu matrix in China. How to fully lyse the cells to release DNA, effectively reduce the residue of Daqu impurities, and obtain high-quality total microbial DNA for molecular biology operations is particularly important.
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Embodiment 1
[0086] Example 1 Extracting Durum Wheat is the DNA of the Luzhou-flavor Daqu Microorganism Produced as Raw Material
[0087] 1. Weigh 1g of Daqu sample, add 0.6g of quartz sand into the mortar (both the mortar and the quartz sand have been sterilized), put the Daqu sample into the mortar and grind it thoroughly, transfer it to a 10mL centrifuge tube, add 4mL of 2 %CTAB extract, and then add 40uL mercaptoethanol;
[0088] 2. Put the EP tube containing the CTAB extract and sample into a 65°C water bath for about 1 hour;
[0089] 3. Take the EP tube out of the water bath, add 20uL proteinase k (20mg / mL), oscillate for 5min to mix, put it in the shaker and continue to oscillate for 30min (temperature is 37°C, speed is 210r / min);
[0090] 4. After oscillating on the shaker for 30 minutes, put the centrifuge tube into the centrifuge, centrifuge at 6000r / min, 4°C for 10 minutes;
[0091] 5. After centrifugation, take 1mL supernatant into a 4mL centrifuge tube, add 500uL equilibrate...
Embodiment 2
[0098] Example 2 Extracting Waxy Wheat as the DNA of Luzhou-flavor Daqu Microorganisms Produced as Raw Materials
[0099] 1. Weigh 1g of Daqu sample, add 0.6g of quartz sand into the mortar (both the mortar and the quartz sand have been sterilized), put the Daqu sample into the mortar and grind it thoroughly, transfer it to a 10mL centrifuge tube, add 4mL of 2 %CTAB extract, and then add 40uL mercaptoethanol;
[0100] 2. Put the EP tube containing the CTAB extract and sample into a 65°C water bath for about 1 hour;
[0101] 3. Take the EP tube out of the water bath, add 20uL proteinase k (20mg / mL), oscillate for 5min to mix, put it in the shaker and continue to oscillate for 30min (temperature is 37°C, speed is 210r / min);
[0102] 4. After oscillating on the shaker for 30 minutes, put the centrifuge tube into the centrifuge, centrifuge at 6000r / min, 4°C for 10 minutes;
[0103] 5. After centrifugation, take 1mL supernatant into a 4mL centrifuge tube, add 500uL equilibrated p...
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Abstract
The invention belongs to the technical field of molecular biology and in particular relates to a method for extracting total microbial DNA from strong-flavor yeasts. The method comprises the following steps: firstly, adding quartz sand into the strong-flavor yeasts for grinding, adding a CTAB solution and mercaptoethanol for water bath treatment, adding proteinase K, and centrifuging; adding Tris-balanced phenol and chloroform isopentyl alcohol into supernatant liquid, and centrifuging; adding chloroform isopentyl alcohol into supernatant liquid, and centrifuging; adding isopropanol into supernatant liquid, freezing, and centrifuging; pouring away upper-layer liquid, washing lower-layer settlings by using ethanol, centrifuging, adding a TE buffer solution into the lower-layer settlings, and storing. The method can be well applied to extraction of total microbial DNA from the strong-flavor yeasts, and the extraction operation is convenient, quick and accurate.
Description
technical field [0001] The invention relates to a method for extracting microbial total DNA from Luzhou-flavor Daqu, belonging to the technical field of molecular biology. Background technique [0002] The use of molecular biology-related methods to study the microbial ecology in the Daqu environment can not only overcome the shortcomings of classical culture methods and the limitations of population loss, but also obtain the colony fingerprints of various microorganisms in environmental samples more reliably and quickly to determine the diversity of microorganisms. The nature and taxonomic status reveal and enrich the microbial resources of the ecological environment. High-quality DNA extraction is the basis of molecular ecology. Whether the DNA in the sample can be quickly and successfully extracted and the quality of the extracted DNA directly affect the success or failure of the entire experiment. Studies have shown that when analyzing microbial communities in a specifi...
Claims
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