CN105483120A - High-temperature combined method for extracting genomic DNA of fish enteric microorganisms - Google Patents
High-temperature combined method for extracting genomic DNA of fish enteric microorganisms Download PDFInfo
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Abstract
The invention discloses a high-temperature combined method for extracting genomic DNA of fish enteric microorganisms. The high-temperature combined method includes the steps that firstly, the walls of various fish enteric microorganisms are properly broken through a lysozyme method and an ultrasonic method at the same time, wherein the ultrasonic physical wall breaking condition ranges from 150 w to 250 w, ultrasonic treatment is carried out for 2-3 s and then carried out again after an interval of 5 s, and the process is repeated 50-60 times; secondly, the sample obtained after ultrasonic treatment and lysozyme are jointly incubated for 30 min in a water-bath constant-temperature oscillator at the temperature of 55-65 DEG C, and the mixture and RNase A are jointly incubated at the temperature of 27-32 DEG C. According to the method, the walls of the various microorganisms are properly broken under the proper and mild ultrasonic condition, and the genomic DNA is prevented from being broken and degraded. As the specific temperature range for incubating the lysozyme and the sample and the specific temperature range for incubating the RNase A and the sample are set, the splitting decomposition effect of the lysozyme can be improved, and it can be avoided that the genomic DNA is degraded by DNA enzymes in tissues and cells. Operation is easy, the extracted genomic DNA is complete and low in degradation rate, and the fish enteric microorganism structure can be truly reflected by the types of the microorganisms identified according to the extracted DNA.
Description
Technical field
The present invention relates to and extract fish intestines microbial genome method, specifically, is a kind of high temperature composite algorithm extracting fish intestines microbe genome DNA.
Background technology
The method extracting microbe genome DNA comprises RNA isolation kit, cetyl trimethylammonium bromide (CTAB) method, lysozyme Method, Physical (as supersonic method, liquid nitrogen grinding method, pearl mill method) etc.Test kit kind market being extracted microbe genome DNA is more, mostly for Mammals, edatope or extracting directly single culture genomic dna, to fish intestines microorganism poor specificity; The DNA fragmentation obtained is little, yield is low, and price is very expensive.
Whether CTAB method has good removal humic acid and the function of other ambient impurities, but only utilizes chemical process broken wall, have broken wall to worry completely; N,O-Diacetylmuramidase can the peptidoglycan of hydrolytic bacteria cell walls effectively, more complete to gram-positive microorganism cracking; And Gram-negative bacteria only has inner wall layer to be peptidoglycan, therefore, N,O-Diacetylmuramidase can not cracking gram-negative bacteria cell wall completely.And although physical method broken wall efficiency is high, needs to grope various condition, comprise power, number of times, time etc., intensity is little just can not broken wall completely; Intensity height can make DNA break even degrade.The method of current extraction animal intestinal microbe genome DNA application is all more single, does not take into account Gram-negative bacteria and gram-positive microorganism and some other kind of quasi-microorganism.In addition, in existing test method, N,O-Diacetylmuramidase and RNaseA work at 37 DEG C of temperature, and in tissue and cell, the lyase such as DNA enzymatic is the most active at this temperature, and DNA can be caused to degrade in a large number.Due to the singularity of fish living environment, its enteric microorganism kind composition is complicated, be subject to the impact of outside environmental elements, existing method is poor to fish intestines microbe genome DNA extraction effect, and the bacterial classification identified is difficult to the composition structure truly reflecting fish intestines microorganism.
Need physics and chemistry method to organically combine so extract fish intestines microbe genome DNA, have complementary advantages, carry out broken wall to the various microorganism of fish intestines, the incubation temperature controlling chemical reagent and sample again farthest avoids the Degradation of DNA enzymatic in tissue and cell.
Summary of the invention
Technical problem to be solved by this invention is, for fish intestines microorganism feature, provides a kind of efficient, economic, high temperature composite algorithm of being applicable to extract fish intestines microbe genome DNA.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is: a kind of high temperature composite algorithm extracting fish intestines microbe genome DNA, it is characterized in that, lysozyme Method after first supersonic method, make fish intestines each monoid microorganism broken wall, the condition of ultrasonic wave physical wall breaking is 150-250w, ultrasonic 2-3s, interval 5s, circulation 50-60 time, sample after ultrasonication and N,O-Diacetylmuramidase are jointly hatched 30min in the water-bath constant temperature oscillator of 55-65 DEG C of temperature, and the temperature of jointly hatching with RNaseA is 27-32 DEG C.
Specifically, comprise the following steps:
(1) get healthy fish enteron aisle, sterile cotton toe-in pricks intestines two ends, and sterile PBS buffer rinses blood, fat in intestinal walls, squeezes out intestinal content, and longitudinally cut open by enteron aisle, scraping intestinal mucosa is translucent to intestinal walls, by the intestinal walls section of being cut into; Enteric contents, mucous membrane and intestinal walls fragment are placed in the centrifuge tube containing PBS, suspension concussion 3 times, each 30s; 4 DEG C subsequently, 800r/min, centrifugal 5min, gets supernatant; 4 DEG C, the centrifugal 5min of 5000r/min; Abandon supernatant, add 1.5mlPBS and repeatedly blow and beat, suspend precipitation, obtains bacterium liquid;
(2) the 2ml centrifuge tube insertion containing 1.5ml bacterium liquid is equipped with in the beaker of trash ice, ultrasonic cell disruption instrument horn is made to be placed in bacterium liquid, under 150-250w condition, ultrasonic 2-3s, interval 5s, circulation 50-60 time, with ruptured microorganism cell, subsequently by bacterium liquid at 4 DEG C, 10000-14000r/min, centrifugal 5min, collecting precipitation;
(3) in precipitation, add the 20mg/ml N,O-Diacetylmuramidase of 50 μ l and 1 × TE damping fluid of 750 μ l, liquid-transfering gun is is softly blown and beaten repeatedly, resuspended precipitation, in water-bath constant temperature oscillator, and incubation 30min under 55-65 DEG C of condition; Be cooled to room temperature, add the 20 μ g/mlRNaseA of 10 μ l, 27-32 DEG C of incubation 30min in water-bath; Add SDS that 100 μ l concentration are 100 μ g/ml, 30 μ l concentration are the Proteinase K of 20mg/ml, reciprocating concussion 2h in 65 DEG C of water-bath constant temperature oscillators;
(4) 1ml phenol chloroform-primary isoamyl alcohol mixed solution is added, phenol chloroform-primary isoamyl alcohol mixed solution phenol by volume: chloroform: primary isoamyl alcohol=25:24:1, puts upside down mixing, centrifugal 2min under 10000-14000r/min condition, get 800 μ l supernatants, move to new centrifuge tube; Add 800 μ l chloroform-isoamyl alcohol mixed solutions subsequently, chloroform-isoamyl alcohol mixed solution chloroform by volume: primary isoamyl alcohol=24:1, puts upside down mixing, centrifugal 5min under 10000-14000r/min condition, gets 600 μ l supernatants, moves to new centrifuge tube; Add the dehydrated alcohol of 3MNaAc and 1.2ml-20 DEG C of precoolings of 60 μ l, be placed in-20 DEG C of refrigerators and precipitate DNA30min; 10000-14000r/min, centrifugal 5min, collecting precipitation;
(5) precipitate 2 times, sucking-off residual alcohol by concentration of volume percent 75% washing with alcohol, room temperature is dried, and adds the 1 × TE damping fluid of 30-50 μ l 50 DEG C of preheatings, dissolving DNA.
Described step (1) is for getting healthy fish, with 75% ethanol body surface of concentration of volume percent, body wall is cut off from anus to backbone direction with blunt nosed scissors, avoid intraperitoneal tissue, scissors bit is chosen, prevents damaged tissue, cut to gill cover trailing edge, cut to lower jaw along gill cover trailing edge again, raise body wall; Isolate enteron aisle with eye scissors, sterile cotton toe-in pricks intestines two ends; Sterile PBS buffer rinses blood, fat in intestinal walls; Squeeze out intestinal content, with eye scissors, enteron aisle is longitudinally cut open, use scalper scraping intestinal mucosa translucent to intestinal walls subsequently, the residue intestinal walls section of being cut into; Enteric contents, mucous membrane and intestines fragment are placed in the centrifuge tube containing PBS, suspension concussion 3 times, each 30s; 4 DEG C subsequently, 800r/min, centrifugal 5min, gets supernatant; 4 DEG C, the centrifugal 5min of 5000r/min; Abandon supernatant, add 1.5mlPBS and repeatedly blow and beat, suspend precipitation, obtains bacterium liquid.
In described step (2), supersonic wave wall breaking condition is: 200W, ultrasonic 2s, interval 5s, circulates 55 times; In described step (3), N,O-Diacetylmuramidase broken wall incubation temperature is 60 DEG C; RNaseA incubation temperature is 30 DEG C.
Described 1 × TE damping fluid is the 0.5MEDTA containing 1MTris-HCl and 0.2ml of 1ml in every 100ml sterilizing deionized water.
The invention has the beneficial effects as follows: step is easy, simple to operate, conventional instrument can complete.Compared with import reagent box, though summary consuming time is of a specified duration, economical and efficient, and the genomic dna obtained is more complete, yield is high; To fish intestines microorganism high specificity, the composition structure that more truly can reflect fish intestines microorganism according to the bacterial classification that identifies of DNA extracted, be applicable to various follow-up test.
Accompanying drawing explanation
Fig. 1 utilizes high temperature composite algorithm (Ultrasonic Conditions: 200w, 2s, interval 5s circulates 55 times; N,O-Diacetylmuramidase incubation temperature: 60 DEG C; RNaseA incubation temperature: 30 DEG C) the bright and beautiful carp enteric microorganism genome dna electrophoresis result (M:Marker that extracts; H: bright and beautiful carp enteric microorganism genomic dna).
Fig. 2 utilizes high temperature composite algorithm (Ultrasonic Conditions: 150w, 3s, interval 5s circulates 60 times; N,O-Diacetylmuramidase incubation temperature: 55 DEG C; RNaseA incubation temperature: 32 DEG C) the bright and beautiful carp enteric microorganism genome dna electrophoresis result (M:Marker that extracts; J: bright and beautiful carp enteric microorganism genomic dna).
Fig. 3 utilizes high temperature composite algorithm (Ultrasonic Conditions: 250w, 2s, interval 5s circulates 60 times; N,O-Diacetylmuramidase incubation temperature: 65 DEG C; RNaseA incubation temperature: 28 DEG C) the Cynoglossus semilaevis enteric microorganism genome dna electrophoresis result (M:Marker that extracts; S: Cynoglossus semilaevis enteric microorganism genomic dna).
Embodiment
Be described in further detail the present invention below in conjunction with the drawings and specific embodiments, described ethanol percentage is volume percent:
The present invention extracts the high temperature composite algorithm of fish intestines microbe genome DNA, comprises the following steps:
1, healthy fish is got, with 75% ethanol body surface.Cut off body wall from anus to backbone direction with blunt nosed scissors, avoid intraperitoneal tissue, scissors bit is chosen, prevent damaged tissue, cut to gill cover trailing edge, then cut to lower jaw along gill cover trailing edge, raise body wall.Isolate enteron aisle with eye scissors, sterile cotton toe-in pricks intestines two ends.Sterile PBS buffer rinses the material such as blood, fat in intestinal walls.
2, squeeze out intestinal content, with eye scissors, enteron aisle is longitudinally cut open, use scalper scraping intestinal mucosa translucent to intestinal walls subsequently, the residue intestinal walls section of being cut into; Enteric contents, mucous membrane and intestines fragment are placed in the centrifuge tube containing PBS, suspension concussion 3 times, each 30s.4 DEG C subsequently, 800r/min, centrifugal 5min, gets supernatant.4 DEG C, the centrifugal 5min of 5000r/min.Abandon supernatant, add 1.5mlPBS and repeatedly blow and beat, suspend precipitation, obtains bacterium liquid.
3, by 75% ethanol ultrasonic cell disruption instrument horn, the 2ml centrifuge tube containing 1.5ml bacterium liquid is inserted and is equipped with in the beaker of trash ice, make horn be placed in bacterium liquid, subsequently beaker is fixed.Under 150-250w condition, ultrasonic 2-3s, interval 5s, circulation 50-60 time, with ruptured microorganism cell.Subsequently by bacterium liquid at 4 DEG C, 10000-14000r/min, centrifugal 5min, collecting precipitation.
4, in precipitation, add the 20mg/ml N,O-Diacetylmuramidase of 50 μ l and 1 × TE damping fluid (containing the 0.5MEDTA of 1MTris-HCl and 0.2ml of 1ml in every 100ml sterilizing deionized water) of 750 μ l, liquid-transfering gun is is softly blown and beaten repeatedly, resuspended precipitation, in water-bath constant temperature oscillator, incubation 30min under 55-65 DEG C of condition.
5, be cooled to room temperature, add the 20 μ g/mlRNaseA of 10 μ l, 27-32 DEG C of incubation 30min in water-bath.
6, the 100 μ g/mlSDS of 100 μ l, the 20mg/ml Proteinase K of 30 μ l is added, 65 DEG C of water-bath constant temperature oscillator reciprocating concussion 2h.
7, add 1ml phenol (25V): chloroform (24V): primary isoamyl alcohol (1V), put upside down mixing, centrifugal 2min under 10000-14000r/min condition, get 800 μ l supernatants, move to new centrifuge tube.Add 800 μ l chloroforms (24V) subsequently: primary isoamyl alcohol (1V), put upside down mixing, centrifugal 5min under 10000-14000r/min condition, get 600 μ l supernatants, move to new centrifuge tube.
8, add the dehydrated alcohol of 3MNaAc and 1.2ml-20 DEG C of precoolings of 60 μ l, be placed in-20 DEG C of refrigerators and precipitate DNA30min.10000-14000r/min, centrifugal 5min, collecting precipitation.
9, precipitate 2 times by 75% washing with alcohol, with low range pipettor sucking-off residual alcohol, room temperature is dried.Add the 1 × TE buffer solution DNA of 30-50 μ l 50 DEG C of preheatings.
First high temperature composite algorithm utilizes PBS suspension ight soil in conjunction with low-speed centrifugal, removes a large amount of soil ulmin that DNA efficiency is extracted in impact.Apply lysozyme Method and supersonic method, be the combination of chemical method and Physical simultaneously.In ultrasonic wave physical wall breaking process, utilize appropriate broken time and the condition of gentleness, make Gram-negative bacteria and gram-positive microorganism and other kind of suitable broken wall of quasi-microorganism, genomic dna can be made again not to be subject to strong physical factor effect to cause fracture, thus improve the yield of DNA, and ensure genomic dna integrity.In high temperature composite algorithm, the application of N,O-Diacetylmuramidase plays a role further for not having the microorganism of complete broken wall after ultrasonication, can improve the lytic effect of N,O-Diacetylmuramidase, solves lysozyme Method to the incomplete defect of gram negative bacterium broken wall simultaneously.This method binding lysozyme method and supersonic method, make Gram-negative bacteria and gram-positive microorganism and other kind of abundant breaking walls and cracking of quasi-microorganism.
The incubation temperature of N,O-Diacetylmuramidase is brought up to about 60 DEG C by high temperature composite algorithm, controls the incubation temperature of RNaseA at about 30 DEG C.Avoid DNA enzymatic degradation of dna in tissue and cell on the one hand, make the genomic dna of extraction more complete, make N,O-Diacetylmuramidase play maximum effect under optimum conditions on the other hand.In order to ensure that N,O-Diacetylmuramidase and Proteinase K fully mix with bacterium liquid in the process of hatching, high temperature composite algorithm uses constant temperature oscillation water-bath in the process of hatching.Eliminate in the process of hatching the process putting upside down mixing, dynamics and speed controlled.
High temperature composite algorithm extract fish intestines microbe genome DNA simple to operate, obtain that genomic dna is complete, degradation rate is low, the microbe species that identifies according to the DNA extracted truly can reflect fish intestines microorganism composition structure.
Embodiment 1
1, healthy bright and beautiful carp is got, with 75% ethanol body surface.Cut off body wall from anus to backbone direction with blunt nosed scissors, avoid intraperitoneal tissue, scissors bit is chosen, prevent damaged tissue, cut to gill cover trailing edge, then cut to lower jaw along gill cover trailing edge, raise body wall.Isolate enteron aisle with eye scissors, sterile cotton toe-in pricks intestines two ends.Sterile PBS buffer rinses the material such as blood, fat in intestinal walls.
2, squeeze out intestinal content, with eye scissors, enteron aisle is longitudinally cut open, use scalper scraping intestinal mucosa translucent to intestinal walls subsequently, the residue intestinal walls section of being cut into; Enteric contents, mucous membrane and intestines fragment are placed in the centrifuge tube containing PBS, suspension concussion 3 times, each 30s.4 DEG C subsequently, 800r/min, centrifugal 5min, gets supernatant.4 DEG C, the centrifugal 5min of 5000r/min.Abandon supernatant, add 1.5mlPBS and repeatedly blow and beat, suspend precipitation, obtains bacteria suspension.
3, by 75% ethanol ultrasonic cell disruption instrument horn, the 2ml centrifuge tube containing 1.5ml bacterium liquid is inserted and is equipped with in the beaker of trash ice, make horn be placed in bacterium liquid, subsequently beaker is fixed.Under 200w condition, ultrasonic 2s, interval 5s, circulate 55 times, with ruptured microorganism cell.Subsequently by bacterium liquid at 4 DEG C, 12000r/min, centrifugal 5min, collecting precipitation.
4, in precipitation, add the 20mg/ml N,O-Diacetylmuramidase of 50 μ l and 1 × TE damping fluid (containing the 0.5MEDTA of 1MTris-HCl and 0.2ml of 1ml in every 100ml sterilizing deionized water) of 750 μ l, liquid-transfering gun is is softly blown and beaten repeatedly, resuspended precipitation, in water-bath constant temperature oscillator, incubation 30min under 60 DEG C of conditions.
5, be cooled to room temperature, add the 20 μ g/mlRNaseA of 10 μ l, 30 DEG C of incubation 30min in water-bath.
6, the 100 μ g/mlSDS of 100 μ l, the 20mg/ml Proteinase K of 30 μ l is added, 65 DEG C of water-bath constant temperature oscillator reciprocating concussion 2h.
7, add 1ml phenol (25V): chloroform (24V): primary isoamyl alcohol (1V), put upside down mixing, centrifugal 2min under 12000r/min condition, get 800 μ l supernatants, move to new centrifuge tube.Add 800 μ l chloroforms (24V) subsequently: primary isoamyl alcohol (1V), put upside down mixing, centrifugal 5min under 12000r/min condition, get 600 μ l supernatants, move to new centrifuge tube.
8, add the dehydrated alcohol of 3MNaAc and 1.2ml-20 DEG C of precoolings of 60 μ l, be placed in-20 DEG C of refrigerators and precipitate DNA30min.12000r/min, centrifugal 5min, collecting precipitation.
9, precipitate 2 times by 75% washing with alcohol, with low range pipettor sucking-off residual alcohol, room temperature is dried.Add the 1 × TE buffer solution DNA of 50 μ l 50 DEG C of preheatings, namely obtain bright and beautiful carp enteric microorganism genomic dna (see Fig. 1).
Embodiment 2
1, healthy bright and beautiful carp is got, with 75% ethanol body surface.Cut off body wall from anus to backbone direction with blunt nosed scissors, avoid intraperitoneal tissue, scissors bit is chosen, prevent damaged tissue, cut to gill cover trailing edge, then cut to lower jaw along gill cover trailing edge, raise body wall.Isolate enteron aisle with eye scissors, sterile cotton toe-in pricks intestines two ends.Sterile PBS buffer rinses the material such as blood, fat in intestinal walls.
2, squeeze out intestinal content, with eye scissors, enteron aisle is longitudinally cut open, use scalper scraping intestinal mucosa translucent to intestinal walls subsequently, the residue intestinal walls section of being cut into; Enteric contents, mucous membrane and intestines fragment are placed in the centrifuge tube containing PBS, suspension concussion 3 times, each 30s.4 DEG C subsequently, 800r/min, centrifugal 5min, gets supernatant.4 DEG C, the centrifugal 5min of 5000r/min.Abandon supernatant, add 1.5mlPBS and repeatedly blow and beat, suspend precipitation, obtains bacteria suspension.
3, by 75% ethanol ultrasonic cell disruption instrument horn, the 2ml centrifuge tube containing 1.5ml bacterium liquid is inserted and is equipped with in the beaker of trash ice, make horn be placed in bacterium liquid, subsequently beaker is fixed.Under 150w condition, ultrasonic 3s, interval 5s, circulate 60 times, with ruptured microorganism cell.Subsequently by bacterium liquid at 4 DEG C, 12000r/min, centrifugal 5min, collecting precipitation.
4, in precipitation, add the 20mg/ml N,O-Diacetylmuramidase of 50 μ l and 1 × TE damping fluid (containing the 0.5MEDTA of 1MTris-HCl and 0.2ml of 1ml in every 100ml sterilizing deionized water) of 750 μ l, liquid-transfering gun is is softly blown and beaten repeatedly, resuspended precipitation, in water-bath constant temperature oscillator, incubation 30min under 55 DEG C of conditions.
5, be cooled to room temperature, add the 20 μ g/mlRNaseA of 10 μ l, 32 DEG C of incubation 30min in water-bath.
6, the 100 μ g/mlSDS of 100 μ l, the 20mg/ml Proteinase K of 30 μ l is added, 65 DEG C of water-bath constant temperature oscillator reciprocating concussion 2h.
7, add 1ml phenol (25V): chloroform (24V): primary isoamyl alcohol (1V), put upside down mixing, centrifugal 2min under 13000r/min condition, get 800 μ l supernatants, move to new centrifuge tube.Add 800 μ l chloroforms (24V) subsequently: primary isoamyl alcohol (1V), put upside down mixing, centrifugal 5min under 13000r/min condition, get 600 μ l supernatants, move to new centrifuge tube.
8, add the dehydrated alcohol of 3MNaAc and 1.2ml-20 DEG C of precoolings of 60 μ l, be placed in-20 DEG C of refrigerators and precipitate DNA30min.13000r/min, centrifugal 5min, collecting precipitation.
9, precipitate 2 times by 75% washing with alcohol, with low range pipettor sucking-off residual alcohol, room temperature is dried.Add the 1 × TE buffer solution DNA of 50 μ l 50 DEG C of preheatings, namely obtain bright and beautiful carp enteric microorganism genomic dna (see Fig. 2).
Embodiment 3
1, healthy Cynoglossus semilaevis is got, with 75% ethanol body surface.Cut off body wall from anus to backbone direction with blunt nosed scissors, avoid intraperitoneal tissue, scissors bit is chosen, prevent damaged tissue, cut to gill cover trailing edge, then cut to lower jaw along gill cover trailing edge, raise body wall.Isolate enteron aisle with eye scissors, sterile cotton toe-in pricks intestines two ends.Sterile PBS buffer rinses the material such as blood, fat in intestinal walls.
2, squeeze out intestinal content, with eye scissors, enteron aisle is longitudinally cut open, use scalper scraping intestinal mucosa translucent to intestinal walls subsequently, the residue intestinal walls section of being cut into; Enteric contents, mucous membrane and intestines fragment are placed in the centrifuge tube containing PBS, suspension concussion 3 times, each 30s.4 DEG C subsequently, 800r/min, centrifugal 5min, gets supernatant.4 DEG C, the centrifugal 5min of 5000r/min.Abandon supernatant, add 1.5mlPBS and repeatedly blow and beat, suspend precipitation, obtains bacteria suspension.
3, by 75% ethanol ultrasonic cell disruption instrument horn, the 2ml centrifuge tube containing 1.5ml bacterium liquid is inserted and is equipped with in the beaker of trash ice, make horn be placed in bacterium liquid, subsequently beaker is fixed.Under 250w condition, ultrasonic 2s, interval 5s, circulate 60 times, with ruptured microorganism cell.Subsequently by bacterium liquid at 4 DEG C, 12000r/min, centrifugal 5min, collecting precipitation.
4, in precipitation, add the 20mg/ml N,O-Diacetylmuramidase of 50 μ l and 1 × TE damping fluid (containing the 0.5MEDTA of 1MTris-HCl and 0.2ml of 1ml in every 100ml sterilizing deionized water) of 750 μ l, liquid-transfering gun is is softly blown and beaten repeatedly, resuspended precipitation, in water-bath constant temperature oscillator, incubation 30min under 65 DEG C of conditions.
5, be cooled to room temperature, add the 20 μ g/mlRNaseA of 10 μ l, 28 DEG C of incubation 30min in water-bath.
6, the 100 μ g/mlSDS of 100 μ l, the 20mg/ml Proteinase K of 30 μ l is added, 65 DEG C of water-bath constant temperature oscillator reciprocating concussion 2h.
7, add 1ml phenol (25V): chloroform (24V): primary isoamyl alcohol (1V), put upside down mixing, centrifugal 2min under 12000r/min condition, get 800 μ l supernatants, move to new centrifuge tube.Add 800 μ l chloroforms (24V) subsequently: primary isoamyl alcohol (1V), put upside down mixing, centrifugal 5min under 12000r/min condition, get 600 μ l supernatants, move to new centrifuge tube.
8, add the dehydrated alcohol of 3MNaAc and 1.2ml-20 DEG C of precoolings of 60 μ l, be placed in-20 DEG C of refrigerators and precipitate DNA30min.12000r/min, centrifugal 5min, collecting precipitation.
9, precipitate 2 times by 75% washing with alcohol, with low range pipettor sucking-off residual alcohol, room temperature is dried.Add the 1 × TE buffer solution DNA of 30 μ l 50 DEG C of preheatings, namely obtain Cynoglossus semilaevis enteric microorganism genomic dna (see Fig. 3).
Above-described embodiment is only for illustration of technological thought of the present invention and feature, its object is to enable those skilled in the art understand content of the present invention and implement according to this, only can not limit the scope of the claims of the present invention with the present embodiment, namely the equal change done of all disclosed spirit or modification, still drop in the scope of the claims of the present invention.
Claims (5)
1. one kind is extracted the high temperature composite algorithm of fish intestines microbe genome DNA, it is characterized in that, lysozyme Method after first supersonic method, make fish intestines each monoid microorganism broken wall, the condition of ultrasonic wave physical wall breaking is 150-250w, ultrasonic 2-3s, interval 5s, circulation 50-60 time, jointly hatches 30min by the sample after ultrasonication and N,O-Diacetylmuramidase in the water-bath constant temperature oscillator of 55-65 DEG C of temperature, and the temperature of jointly hatching with RNaseA is 27-32 DEG C.
2. the high temperature composite algorithm of extraction fish intestines microbe genome DNA according to claim 1, is characterized in that, comprise the following steps:
(1) get healthy fish enteron aisle, sterile cotton toe-in pricks intestines two ends, and sterile PBS buffer rinses blood, fat in intestinal walls, squeezes out intestinal content, and longitudinally cut open by enteron aisle, scraping intestinal mucosa is translucent to intestinal walls, by the intestinal walls section of being cut into; Enteric contents, mucous membrane and intestinal walls fragment are placed in the centrifuge tube containing PBS, suspension concussion 3 times, each 30s; 4 DEG C subsequently, 800r/min, centrifugal 5min, gets supernatant; 4 DEG C, the centrifugal 5min of 5000r/min; Abandon supernatant, add 1.5mlPBS and repeatedly blow and beat, suspend precipitation, obtains bacterium liquid;
(2) the 2ml centrifuge tube insertion containing 1.5ml bacterium liquid is equipped with in the beaker of trash ice, ultrasonic cell disruption instrument horn is made to be placed in bacterium liquid, under 150-250w condition, ultrasonic 2-3s, interval 5s, circulation 50-60 time, with ruptured microorganism cell, subsequently by bacterium liquid at 4 DEG C, 10000-14000r/min, centrifugal 5min, collecting precipitation;
(3) in precipitation, add the 20mg/ml N,O-Diacetylmuramidase of 50 μ l and 1 × TE damping fluid of 750 μ l, liquid-transfering gun is is softly blown and beaten repeatedly, resuspended precipitation, in water-bath constant temperature oscillator, and incubation 30min under 55-65 DEG C of condition; Be cooled to room temperature, add the 20 μ g/mlRNaseA of 10 μ l, 27-32 DEG C of incubation 30min in water-bath; Add SDS that 100 μ l concentration are 100 μ g/ml, 30 μ l concentration are the Proteinase K of 20mg/ml, reciprocating concussion 2h in 65 DEG C of water-bath constant temperature oscillators;
(4) 1ml phenol chloroform-primary isoamyl alcohol mixed solution is added, phenol chloroform-primary isoamyl alcohol mixed solution phenol by volume: chloroform: primary isoamyl alcohol=25:24:1, puts upside down mixing, centrifugal 2min under 10000-14000r/min condition, get 800 μ l supernatants, move to new centrifuge tube; Add 800 μ l chloroform-isoamyl alcohol mixed solutions subsequently, chloroform-isoamyl alcohol mixed solution chloroform by volume: primary isoamyl alcohol=24:1, puts upside down mixing, centrifugal 5min under 10000-14000r/min condition, gets 600 μ l supernatants, moves to new centrifuge tube; Add the dehydrated alcohol of 3MNaAc and 1.2ml-20 DEG C of precoolings of 60 μ l, be placed in-20 DEG C of refrigerators and precipitate DNA30min; 10000-14000r/min, centrifugal 5min, collecting precipitation;
(5) precipitate 2 times, sucking-off residual alcohol by concentration of volume percent 75% washing with alcohol, room temperature is dried, and adds the 1 × TE damping fluid of 30-50 μ l 50 DEG C of preheatings, dissolving DNA.
3. the high temperature composite algorithm of extraction fish intestines microbe genome DNA according to claim 2, it is characterized in that, described step (1), for getting healthy fish, with 75% ethanol body surface of concentration of volume percent, cuts off body wall from anus to backbone direction with blunt nosed scissors, avoid intraperitoneal tissue, scissors bit is chosen, prevents damaged tissue, cut to gill cover trailing edge, cut to lower jaw along gill cover trailing edge again, raise body wall; Isolate enteron aisle with eye scissors, sterile cotton toe-in pricks intestines two ends; Sterile PBS buffer rinses blood, fat in intestinal walls; Squeeze out intestinal content, with eye scissors, enteron aisle is longitudinally cut open, use scalper scraping intestinal mucosa translucent to intestinal walls subsequently, the residue intestinal walls section of being cut into; Enteric contents, mucous membrane and intestines fragment are placed in the centrifuge tube containing PBS, suspension concussion 3 times, each 30s; 4 DEG C subsequently, 800r/min, centrifugal 5min, gets supernatant; 4 DEG C, the centrifugal 5min of 5000r/min; Abandon supernatant, add 1.5mlPBS and repeatedly blow and beat, suspend precipitation, obtains bacterium liquid.
4. the high temperature composite algorithm of extraction fish intestines microbe genome DNA according to claim 2, is characterized in that, in described step (2), supersonic wave wall breaking condition is: 200W, ultrasonic 2s, interval 5s, circulates 55 times; In described step (3), N,O-Diacetylmuramidase broken wall incubation temperature is 60 DEG C; RNaseA incubation temperature is 30 DEG C.
5. the high temperature composite algorithm of extraction fish intestines microbe genome DNA according to claim 2, is characterized in that, described 1 × TE damping fluid is the 0.5MEDTA containing 1MTris-HCl and 0.2ml of 1ml in every 100ml sterilizing deionized water.
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