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A rapid algal DNA extraction method

An extraction method, algae technology, which is applied in the field of rapid algae DNA extraction, can solve the problems of protein pollution and low purity, and achieve the effect of short extraction time, simple extraction steps, and simplified sample pretreatment and extraction process

Active Publication Date: 2021-06-29
李润成
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the concentration of extracted DNA is 0.29 μg / μL, and the A260 / A280 is 1.546. Although the concentration of extracted genomic DNA is high, the purity is low and there is protein contamination, which can be used for related molecular biology research, but a small amount of protein Whether it exists or has a certain influence on the research

Method used

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  • A rapid algal DNA extraction method
  • A rapid algal DNA extraction method
  • A rapid algal DNA extraction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Extraction of algae genomic DNA

[0041] 1. Reagent of the present invention:

[0042] Nuclease-free water, manufacturer: Ambion, item number: AM9932

[0043] BSA, Manufacturer: NEB, Item No.: B9001

[0044] 2, main experimental instrument of the present invention:

[0045] Eppendorf High Speed ​​Refrigerated Centrifuge

[0046] 3. Gel imaging system, manufacturer: Shanghai Qinxiang Scientific Instrument Co., Ltd.

[0047] 4. Extraction of algae genomic DNA

[0048] Collect 1 L of algae-rich lake water samples from different locations, filter with a 0.22 μm filter membrane, enrich the algae samples, and extract algae genomic DNA according to the following scheme:

[0049] 1) Filter and collect 50mg of algae sample into a 1.5mL centrifuge tube, add 100μL Buffer A, 500μL sterile water, mix well; add 400μL Buffer B, vortex and mix for 30s; then add 300μL Buffer C, vortex and mix 30s; centrifuge at 8000g for 2 minutes (low-speed centrifugation is often repr...

Embodiment 2

[0064] Example 2: Microcystis 16S rDNA PCR amplification

[0065] 1. Reagent of the present invention:

[0066] Nuclease-free water, manufacturer: Ambion, item number: AM9932

[0067] BSA, Manufacturer: NEB, Item No.: B9001

[0068] PCR amplification reagents, manufacturer: Treasure Biotechnology Co., Ltd., item number: R045Q

[0069] 2. Experimental instrument of the present invention:

[0070] Eppendorf High Speed ​​Refrigerated Centrifuge

[0071] 3. Gel imaging system, manufacturer: Shanghai Qinxiang Scientific Instrument Co., Ltd.

[0072] 4. Microcystis 16S rDNA PCR amplification

[0073] 16S rDNA PCR amplification of the algae genome was carried out using primers in the V3 region of 16s. The primer sequence 16S-F: CCTACGGGNGGCWGCAG, 16S-R: GACTACHVGGGGTATCTAATCC, the size of the amplified target fragment was about 318bp.

[0074] The PCR reaction system was as follows: 1 μL of 10 μM 16s-F and 16s-R primers; 2.5 μL of 10×PCR buffer; 2 μL of 25 μM dNTP; 1.5 μL of 15...

Embodiment 3

[0078] Example 3: Fluorescent quantitative PCR detection of toxin-producing Microcystis FACHB-315

[0079] 1. Reagent of the present invention:

[0080] Nuclease-free water, manufacturer: Ambion, item number: AM9932

[0081] BSA, Manufacturer: NEB, Item No.: B9001

[0082] Fluorescence quantitative PCR amplification reagent, manufacturer: Treasure Biotechnology Co., Ltd., item number: DRR041S

[0083] 2. Experimental instrument of the present invention:

[0084] Eppendorf High Speed ​​Refrigerated Centrifuge

[0085] Fluorescence quantitative PCR instrument, manufacturer model: Thermofisher ABI7500 quantitative PCR instrument

[0086] 3. Fluorescent quantitative PCR detection

[0087] Using microcystin toxin-producing gene mcyE / ndaF primers for PCR amplification,

[0088] Primer sequence mcy-F: AATAAATCATAATTTAGAACSGGVGATTTAGG;

[0089] mcy-R: AATAAATCATAACGRBTVADTTGRTATTCAATTTCT, the size of the amplified target fragment is about 128bp.

[0090] The PCR reaction syste...

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Abstract

The invention discloses a rapid algal DNA extraction method, which relates to the technical field of biological DNA extraction, and can efficiently and rapidly extract the DNA of various algae samples, so that the extracted DNA has high concentration and high purity, and can be directly extracted. For follow-up research and testing. Collect 50mg of algae samples by filtration into a 1.5mL centrifuge tube, add BufferA, BufferB and BufferC in sequence, and shake and mix separately; Use phenol chloroform isoamyl alcohol to remove protein, add isopropanol solution to vibrate and mix, add to silicon matrix membrane nucleic acid purification column and centrifuge, discard waste liquid and rinse, rinse and centrifuge again to dry, add eluent to obtain algae genomic DNA solution.

Description

technical field [0001] The invention relates to the technical field of DNA extraction of organisms, in particular to a rapid algae DNA extraction method. Background technique [0002] Metagenomics has brought unprecedented opportunities to maximize the mining of microbial resources, and has become the most important hotspot and frontier of international life science and technology research and development. In order to fully understand the rich resources of non-culturable microorganisms, combined with the development of modern molecular biology techniques, many new methods and technologies that do not require independent cultivation of microorganisms have been established. Such as DGGE, 16s sequencing, metagenomic sequencing, etc. Rapid and high-quality environmental DNA acquisition is an important prerequisite for the realization of the above research, so it is necessary to conduct research on environmental DNA extraction methods. [0003] Algae is currently considered to ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/101C12Q1/6806C12Q2531/113C12Q2549/125
Inventor 李润成
Owner 李润成