CN103374566A - Efficient method for extracting purified total DNA (deoxyribonucleic acid) from lake sediment sample - Google Patents

Abstract

The invention discloses an efficient method for quickly extracting purified total DNA (deoxyribonucleic acid) from a lake sediment sample. The method comprises the following steps of: (1) putting about 0.3-1.0g of a sample into a 2mL centrifuge tube containing a plurality of 2.5mm sterilization glass beads, adding 300-1000 microliters of a DNA extraction buffer solution, then adding 50-500 microliters of a potassium dichromate solution, eddying for 10-60 seconds by using Vortex-2 (Mobio Laboratories Inc), adding 50-300 microliters of an SDS (sodium dodecyl sulfate) solution, eddying for 3 minutes, and incubating for 5-60 minutes at 65 DEG C; and (2) adding 50-200 microliters of a potassium chloride solution, and severely bottoming up and uniformly mixing up and down for a plurality of times. According to the method, the total DNA of sediment soil can be quickly and efficiently extracted, and meanwhile, special effects of removing corrosion and deodorizing are achieved, so that the technical difficulties during extraction of DNA from an eutrophication water body sediment are solved, and high-purity DNA test samples are provided for meta-genome researches of environmental samples including soil, sludge, sediments, mud and the like and various kinds of gene amplification.

Description

A kind of high efficiency method that from the lacustrine deposit matter sample, extracts the total DNA of purifying

Technical field

The present invention relates to a kind of efficiently Sediment environment sample total DNA extracting method fast.

Background technology

The Metagenomics DNA extraction is the basis of metagenomics research.At present, more for soil DNA extracting method, such as liquid nitrogen grinding method, broken microwave method, granulated glass sphere cracking process, enzymatic lysis method, rapid frozen thaw method, ion exchange method, solvent-granulated glass sphere mode of averaging, SDS-glass bead method, SDS-GITC-PEG method, MS method, Nycodenz method, Blending method, UltraClean Soil DNA kit (MoBio Laboratories, Inc., Solana Beach, Calif.), Fast DNA spin sample kit (for soil; Bio 101, Lajolla, Calif.), and calcium chloride-SDS-enzyme process and granulated glass sphere-calcium chloride-SDS method etc.For the eutrophic lake sediment sample, designed special soil DNA extraction novel method on this basis.

Summary of the invention

The invention provides a kind of efficiently Sediment environment sample total DNA extracting method fast.

Removing humic acid is the important step of soil total DNA extraction, and step 1 can efficiently be removed humic acid, improves settling soil total DNA extraction purity.

Humic acid in the efficient removal settling soil improves total DNA extraction purity, learns research for soil metagenome basic technology is provided.

Description of drawings

Figure 1A-1 extracts 3 kinds of different settling soil microbe genome DNA electrophorograms (3 repetitions) for the inventive method;

Figure 1A-2 extracts 3 kinds of different settling microbial genome 16S rDNA gene fragment amplification electrophorograms for the inventive method;

Figure 1A-3 extracts 3 kinds of different settling microorganism ammonia oxidation archaeal genome amoA gene fragment amplification electrophorograms for the inventive method.

Figure 1A-4 is that the inventive method is extracted 3 kinds of different settling microorganism ammonia oxidizing bacteria genome amoA gene fragment amplification electrophorograms with A-5;

Figure 1A-6 extracts 3 kinds of different settling microorganism methane-oxidizing bacteria genome pmoA gene fragment amplification electrophorograms for the inventive method.

Figure 1A-7 is that the inventive method is extracted 3 kinds of different settling microorganism denitrifying bacteria genome nosZ gene fragment amplification electrophorograms with A-8;

Figure 1A-9 extracts 3 kinds of different settling microorganism vinelandii genome nifH gene fragment amplification electrophorograms for the inventive method.

Embodiment

Below in conjunction with specific embodiment, the present invention is described in detail.

Experiment soil: be collected in Inner Mongol Ulansuhai Nur eutrophication sediment sample for the examination soil sample, be designated as respectively WL1, WL2, WL3.Sampling depth: 0-20cm, the part physico-chemical property of 3 kinds of pedotheques sees Table 1.

Efficient lake sediment environmental sample total DNA extraction method, operation steps is as follows:

(1) gets 0.3g-1.0g left and right sides settling soil sample in the centrifuge tube of the 2mL that three 2.5mm sterilization granulated glass spherees are housed, 300-1000 μ L DNA extraction damping fluid (100mmol/L Tris-HCl, 1.5mol/L NaCl, 1%CTAB, pH 8.0), add again 50-500 μ L potassium bichromate solution (0.05mol/L-3mol/L), use Vortex-

2 (Mobio Laboratories Inc) vortex 10s-1min adds 50-300 μ L SDS solution (5%-20%), vortex 3min, 65 ℃ of incubation 5-60min;

(2) add 50-200 μ L Klorvess Liquid (0.05mol/L-5mol/L), acutely put upside down up and down mixing for several times, the centrifugal 2min of 12000r/min;

(3) get supernatant 1000 μ L in the 2mL centrifuge tube, add isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1), the centrifugal 5min of 12000r/min;

(4) get supernatant 900 μ L in the 1.5mL centrifuge tube, add the Virahol of 0.6 times of volume, ice bath 10min, the centrifugal 5min of 12000r/min;

(5) abandon supernatant, wash precipitation with 70% alcohol, seasoning or lyophilize;

(6) 50 μ L TE dissolution precipitations.

Experimental result: see Figure 1A-1, can find out that from Figure 1A-1 the Sediment environment genomic dna size that this method extraction obtains is about about 23kb.

Soil total DNA extraction quality verification: pcr amplification is the final purpose of settling DNA extraction, therefore, can carry out pcr amplification and become a most important index of settling DNA extraction quality, the result sees that Figure 1A-2 is to A-9, can find out that from Figure 1A-2 to A-9 the settling genomic dna that this method obtains can be used for 16S rDNA, the ancient bacterium functional gene of ammonia oxidation, ammonia oxidizing bacteria, the methane-oxidizing bacteria functional gene, the amplification of denitrifying bacteria functional gene and vinelandii functional gene.

Conclusion: method provided by the invention can obtain the total DNA of highly purified settling soil, can be successfully applied to follow-up molecular biological analysis, for the research of settling edatope metagenomics provides a kind of new total DNA extraction technology.

Claims (3)

1. a Sediment environment sample total DNA rapid extracting method is characterized in that, may further comprise the steps:

(1) gets 0.3g-1.0g left and right sides settling soil sample in the centrifuge tube of the 2mL that three 2.5mm sterilization granulated glass spherees are housed, 300-1000 μ LDNA Extraction buffer (100mmol/L Tris-HCl, 1.5mol/L NaCl, 1%CTAB, pH 8.0), add again 50-500 μ L potassium bichromate solution (0.05mol/L-3mol/L), use Vortex-

2 (Mobio Laboratories Inc) vortex 10s-1min adds 50-300 μ LSDS solution (5%-20%), vortex 3min, 65 ℃ of incubation 5-60min;

(2) add 50-200 μ L Klorvess Liquid (0.05mol/L-5mol/L), acutely put upside down up and down mixing for several times, the centrifugal 2min of 12000r/min;

(3) get supernatant 1000 μ L in the 2mL centrifuge tube, add isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1), the centrifugal 5min of 12000r/min;

(4) get supernatant 900 μ L in the 1.5mL centrifuge tube, add the Virahol of 0.6 times of volume, ice bath 10min, the centrifugal 5min of 12000r/min;

(5) abandon supernatant, wash precipitation with 70% alcohol, seasoning or lyophilize;

(6) 50 μ LTE dissolution precipitations.

2. potassium bichromate solution (0.05mol/L-3mol/L) in the step 1.

3. Klorvess Liquid (0.05mol/L-5mol/L) in the step 2.

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