A method for extracting microbial genome dna from poplar wood

A technology of microorganisms and genomes, applied in the field of molecular biology, can solve problems such as pollution, unsatisfactory, and low average concentration of DNA, and achieve the effects of high concentration, reduced degradation and damage, and increased lysing ability and cleavage degree

CN106754872BActive Publication Date: 2019-07-05HUAZHONG AGRI UNIV +1
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Publication Date
2019-07-05

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Abstract

The invention discloses a method for extracting microbial genome DNA from poplar wood. According to the method, a CTAB (cetyltrimethyl ammonium bromide) method and an SDS (sodium dodecyl sulfonate) method are combined, and technical parameters of various steps are optimized, so that the genome DNA extracted by virtue of the method has the advantages of being high in concentration and free from pollution; determined by a spectrophotometer, OD260 / OD230 and OD260 / OD280 values are close to standard values, completely satisfying requirements of 16S rDNA / ITS sequencing and metagenomics sequencing adopted in the later period; therefore, the method can be directly applied to a molecular operation.
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Description

technical field

[0001] The invention belongs to the technical field of molecular biology, in particular to a method for extracting microbial genome DNA from poplar wood (divided into wet heartwood and healthy sapwood) applied in molecular sequencing technology. Background technique

[0002] Poplar is one of the important fast-growing timber species in my country. The seventh national forest resources inventory data show that the total area of ​​poplar forests in my country has reached 10.1026 million hm 2 , of which the artificial forest is 7.5723 million hm 2 , accounting for 18.9% of the total area of ​​artificial arbor forests in the country, and the cultivated area ranks first in the world. However, the phenomenon of wet heartwood is very serious, which has a negative impact on the processing and utilization of poplar. According to a survey in Jiayu County, Hubei Province, the rate of diseased plants in nine poplar varieties reached 100%, the discoloration diameter at...

Examples

Embodiment 1

[0031] The method provided by the present invention is used below to extract microbial genomic DNA from poplar wet heartwood and healthy sapwood samples, and each treatment is repeated 3 times.

[0032] Experimental operation:

[0033] 1. Preparation of extraction buffer and TE buffer:

[0034] Extraction buffer: 1%CTAB+2%PVPP+1.5M NaCl+100mM EDTA+100mM TE pH8.0+0.1M sodium phosphate buffer pH8.0, autoclaved.

[0035] TE buffer: 10 mM Tris-HCl and 1 mM EDTA.

[0036] 2. Extraction step

[0037] (1) Grinding the sample with liquid nitrogen.

[0038] (2) Take 1g of ground wet heartwood or healthy sapwood sample, add 0.6g of activated carbon, add 5mL of extraction buffer (1%CTAB+2%PVPP+1.5M NaCl+100mM EDTA+100mM TE pH8.0+0.1M Sodium phosphate buffer (pH8.0), add 75 μL proteinase K (20 mg / ml), vortex mix, add 1000 μL SDS (10%), vortex mix.

[0039] (3) Place in a 60°C water bath for 2 hours, during which time mix by inverting 3 to 4 times.

[0040] (4) Centrifuge (12000rpm, ...