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Reagent case for extracting zooxanthellae genom from ledge rock coral and method thereof

A technology of zooxanthellae and genome, which is applied to the kit for extracting zooxanthellae genomic DNA from reef-building corals and its field, which can solve the problem of increasing the number of extracted high-quality zooxanthellae genome DNA, having organic solvents, and cumbersome methods, etc. problem, to achieve the effect of fast extraction method, simple operation, and comprehensive genetic information

Inactive Publication Date: 2011-01-19
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because zooxanthellae live symbiotically in reef-building corals, and reef-building corals themselves have calcium carbonate skeletons, calcium carbonate salts will mechanically shear genomic DNA fragments during the extraction process, thereby increasing the extraction of high-quality zooxanthellae genomic DNA. Difficulty
However, the conventional phenol / chloroform extraction method is not suitable for routine detection due to the cumbersome method and the toxicity of organic solvents.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Make the Zooxanthellae Genomic DNA Extraction Kit according to the following formula:

[0032] Reagent A: pH 8.0, 250mM Tris-Boric solution, 50mM EDTA solution, 2% SDS solution, 100mM NaCl solution and distilled water in a volume ratio of 25:10:10:2:53 ;

[0033] Reagent B: Protease E, its concentration is 10mg / ml;

[0034] Reagent C: RNAase A, its concentration is 20mg / ml;

[0035] Reagent D: supersaturated NaCl solution containing 7M NaCl;

[0036] Reagent E: DNA washing solution, ethanol solution with a volume concentration of 75%;

[0037] Reagent F: DNA eluent, pH 8.0, 10mM TE buffer.

[0038] DNA adsorption column: silica gel column.

[0039] The following procedure was followed to extract the zooxanthellae genomic DNA of the beautiful staghorn coral symbiosis:

[0040] (1) Sample processing: take about 30mg of beautiful staghorn coral, add liquid nitrogen to fully grind it into powder, and carefully pour it into a 2ml centrifuge tube;

[0041] (2) Cell lys...

Embodiment 2

[0047] Make the Zooxanthellae Genomic DNA Extraction Kit according to the following formula:

[0048] Reagent A: pH 8.0, 100mM Tris-Boric solution, 10mM EDTA solution, 1% SDS solution, 50mM NaCl solution and distilled water in a volume ratio of 25:10:10:2:53 ;

[0049] Reagent B: Protease E, its concentration is 5mg / ml;

[0050] Reagent C: RNAase A, its concentration is 5mg / ml;

[0051] Reagent D: supersaturated NaCl solution containing 5M NaCl;

[0052] Reagent E: a DNA washing solution, which is an ethanol solution with a volume concentration of 60%;

[0053] Reagent F: DNA eluent, pH 8.0, 5mM TE buffer.

[0054] DNA adsorption column: silica gel column.

[0055] The following procedure was followed to extract the zooxanthellae genomic DNA of the symbiosis of the chrysanthemum coral:

[0056] (1) Sample treatment: Take about 10mg of reef-building coral, add liquid nitrogen to fully grind it into powder, and carefully pour it into a 2ml centrifuge tube;

[0057] (2) Ce...

Embodiment 3

[0063] Make the Zooxanthellae Genomic DNA Extraction Kit according to the following formula:

[0064] (a) Reagent A: by pH8.0, the Tris-Boric solution of 300mM, the EDTA solution of 100mM, the SDS solution of mass concentration 5%, the NaCl solution of 200mM and distilled water press the volume ratio of 25:10:10:2:53 mixed;

[0065] (b) Reagent B: protease E, its concentration is 15mg / ml;

[0066] (c) Reagent C: RNAase A, its concentration is 30mg / ml;

[0067] (d) Reagent D: a supersaturated NaCl solution containing 7M NaCl;

[0068] (e) Reagent E: a DNA washing solution, which is an ethanol solution with a volume concentration of 80%;

[0069] (f) Reagent F: DNA eluent, pH 8.0, 20 mM TE buffer.

[0070] DNA adsorption column: silica gel column.

[0071] The following procedure was followed to extract the zooxanthellae genomic DNA of the symbiosis of the chrysanthemum coral:

[0072] (1) Sample treatment: Take about 30mg of reef-building coral, add liquid nitrogen to ful...

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Abstract

The invention provides a reagent kit which extracts zooxanthella genome DNA from reef building coral and a method of the reagent kit. The reagent kit comprises: reagent A (lysis buffer), reagent B (protease E), reagent C (RNAase A), reagent D (supersaturation NaC1 solution), reagent E (DNA cleaning mixture) and reagent F (DNA eluent). The method of the invention comprises: firstly, using the lysis buffer which contains SDS to crack tissue cells, then, providing a high salt low PH circumstance, enabling protein in solution to degenerate further, but combining DNA on a silica gel column, and finally, quantitatively reclaiming the DNA from a chromatography column in water-soluble buffer solution. The zooxanthella genome DNA can be rapidly and conveniently extracted from varous reef building coral, the DNA which is obtained can be used to research molecular ecology, systematic evolution and the like, and thereby the demands of regular monitoring tasks in coral reef nature reserves can be satisfied.

Description

technical field [0001] The invention relates to a kit for extracting zooxanthellae genome DNA from reef-building stony corals and a method for extracting reef-building stony coral symbiotic zooxanthellae genome DNA using the kit. Background technique [0002] Coral reefs have extremely high biodiversity. 25% of the world's marine life lives in coral reefs. They are known as "Oasis of the Sea" or "Marine Tropical Rainforest", and their socio-economic value is immeasurable. The coral reef ecosystem has extremely rich biological resources and is a breeding base for fisheries, providing human beings with important food and medicine resources; the coral reefs on the seabed have beautiful scenery and are famous underwater gardens. They are an excellent diving tourist destination for people. Therefore, the protection and sustainable use of coral reefs has attracted people's attention. For example, my country has established the Sanya Coral Reef National Nature Reserve in Hainan and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
Inventor 黄晖董志军黄良民李元超
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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