CN102936592A - Fast and high-efficiency extracting and purifying method of corn mitochondrial DNA (deoxyribonucleic acid) - Google Patents

Abstract

The invention discloses a fast and high-efficiency extracting and purifying method of corn mitochondrial DNA (deoxyribonucleic acid). The extracting and purifying method comprises the steps of charging corn explants into a pre-cooled mortar in a splitting manner, adding 4mL of pre-cooled extraction buffer solution A in 1g of sample, adding 1-2g of quartz sands, and grinding to be a homogenate; taking three 10mL centrifugal tubes, respectively numbering the centrifugal tubes as A, B and C, then taking three 2mL centrifugal tubes and respectively numbering the centrifugal tubes as D, E and F; and in the extraction process, processing with RNase A and RNase T1 simultaneously to completely remove RNA (ribonucleic acid) pollution. MgCl2 has the function of DNaseI buffer so as to be capable of replacing DNaseI buffer, and the cost is lowered. Due to the adoption of the method disclosed by the invention, the output rate of mtDNA is improved obviously.

Description

Extraction and the purification process of a kind of rapidly and efficiently Corn Mitochondria DNA

Technical field

What the present invention relates to is extraction and the purification process of a kind of rapidly and efficiently Corn Mitochondria DNA.

Background technology

Plant mtDNA extracting method mainly contains the methods such as density gradient centrifugation, differential centrifugation; Though the Mitochondrial DNA purity that density gradient centrifugation obtains is high, complicated operation, consuming time, and also large to the demand of material, the mtDNA productive rate is low simultaneously, and long-time high speed centrifugation also requires very high to whizzer; Utilize density gradient centrifugation to extract the Mitochondrial DNA experimental result as shown in Figure 1, its master tape is unintelligible, and the band disperse is seriously polluted, and loaded down with trivial details, the consuming time length of process; Although common differential centrifugation is simple to operate, low, the easy degraded of the mtDNA purity of extracting, and the contaminating impurities such as nuclear DNA, RNA and protein are serious, can not satisfy the follow-up study requirements of one's work; High salt-Proteinase K method extraction Mitochondrial DNA has simple to operate, and the mtDNA that extracts has structural integrity, purity and productive rate than advantages of higher, can satisfy the needs of pcr analysis, the assignment of genes gene mapping, the operation of clone's equimolecular.

Summary of the invention

Technical problem to be solved by this invention is extraction and the purification process that a kind of rapidly and efficiently Corn Mitochondria DNA is provided for the deficiencies in the prior art.

Technical scheme of the present invention is as follows:

Extraction and the purification process of a kind of rapidly and efficiently Corn Mitochondria DNA may further comprise the steps:

(1) the corn explant is divided in the mortar that is filled to precooling, add the Extraction buffer A of precooling in the ratio of every gram sample 4mL, put into 1-2g quartz sand, be ground to homogenate; The centrifuge tube of getting 3 10mL is numbered respectively A, B, C, and the centrifuge tube of getting again 3 2mL is numbered respectively D, E, F.

(2) filter homogenate with 6 layers of sterile gauze, will grind tissue and put back in the mortar, add buffer A by every gram sample 2mL and again grind and filter, 2 times filtrate merges.To install to filtrate dividing in the A pipe, the centrifugal 10min of 500r/min removes large stretch of fragment tissue;

(3) supernatant liquor is transferred in the B pipe, centrifugal 2 times of 2600r/min is respectively 15 and 10min, removes the plastids such as chloroplast(id);

(4) get supernatant liquor to the C pipe, centrifugal 15 min of 10000 r/min abandon supernatant, add the A liquid of 1 mL precooling in precipitation, blow afloat gently precipitation, mixing with the rifle head.The centrifugal 10min of 1000r/min sucks supernatant in the D pipe, adds the 100mg/mL DNaseI of 1/500 times of volume and the 1mol/L MgCl of 1/100 times of volume ₂The 0.5mol/L Na that adds 1/25 times of volume behind the ice bath 1h ₂EDTA leaves standstill 10 min, stops the DNaseI reaction;

(5) the centrifugal 15min of 13200r/min removes supernatant, obtains the plastosome precipitation, adds ST liquid purifying 1 time again, obtains the plastosome of purifying;

(6) add the 1mL lysate B precipitation that suspends in the D pipe precipitation, add again 25mg/mL Proteinase K 2.5 μ L, 50 ℃ of incubation 1 h, 37 ℃ of incubation 1h again, during the interval shake;

(7) take out the centrifuge tube room temperature and place, after its cooling, add isopyknic phenol: chloroform: the primary isoamyl alcohol mixed solution, mixed solution mixed volume ratio is 25:24:1, fully mixing is placed on and shakes 15min on the shaking table, centrifugal 10 min of 10000 r/min;

(8) turn supernatant liquor to the E pipe, add isopyknic chloroform: the primary isoamyl alcohol mixed solution, mixed solution mixed volume ratio is 24:1, turns upside down to shake up approximately 10min centrifugal 10 min of 10000 r/min;

(9) supernatant liquor is transferred in the F pipe, then adds the RNaseT of the 1000U/ μ L of the RNaseA of 1/100 volume 10mg/mL and 1/500 volume ₁, RNA is removed in 37 ℃ of water-baths 1 hour;

(10) add isopyknic chloroform: the primary isoamyl alcohol mixed solution, mixed solution mixed volume ratio 24:1, turning upside down shakes up approximately 10min, centrifugal 10 min of 10000 r/min;

(11) get 0. 1 times of volume NaAc and 2 times of volume dehydrated alcohols that supernatant liquor adds precooling to the centrifuge tube of 1.5mL ,-20 ℃ are spent the night;

(12) 4 ℃, the centrifugal 15min of 13200r/min abandon supernatant, and precipitation is with 75% washing with alcohol 2 ~ 3 times;

(13) dissolve in a small amount of water after being deposited in air drying ,-20 ℃ of refrigerations are for subsequent use.

Used reagent wherein:

(1) buffer A: 1mol/L Tris-HCl 12.5mL, 0.5mol/L Na ₂EDTA 12.5mL, 1.3 mol/L NaCl 15.743g are settled to 250mL with the sterilization distilled water, autoclave sterilization, room temperature preservation.Add 0.5g BSA, 0.125g halfcystine, 750 μ L beta-mercaptoethanols before using.

(2) ST liquid: sucrose 13.6916g, 1mol/L Tris-HCl 5mL, 0.5mol/L Na ₂EDTA 5mL is settled to 100mL with the sterilization distilled water, autoclave sterilization, room temperature preservation.Add 0.1g BSA before using.

(3) lysate B:1mol/L Tris-HCl 2.5mL, 0.5mol/L Na ₂EDTA 4mL, 10% SDS 5mL is settled to 100mL with the sterilization distilled water, room temperature preservation.

Beneficial effect of the present invention:

(1) this experiment is adopted two kinds of RNA enzyme RNase A and RNase T in leaching process ₁Process simultaneously the pollution that can remove RNA fully.

(2) MgCl ₂Effect with DNaseI buffer, alternative DNaseI buffer reduces cost.

(3) output capacity of mtDNA is improved significantly.

Description of drawings

Fig. 1 is for utilizing density gradient centrifugation to extract the Mitochondrial DNA experimental result;

Fig. 2 is for adopting method of the present invention to extract the Mitochondrial DNA experimental result;

Embodiment

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

(1) the corn explant is divided in the mortar that is filled to precooling, add the Extraction buffer A of precooling in the ratio of every gram sample 4mL, put into 1-2g quartz sand, be ground to homogenate; The centrifuge tube of getting 3 10mL is numbered respectively A, B, C, and the centrifuge tube of getting again 3 2mL is numbered respectively D, E, F.

(2) filter homogenate with 6 layers of sterile gauze, will grind tissue and put back in the mortar, add buffer A by every gram sample 2mL and again grind and filter, 2 times filtrate merges.To install to filtrate dividing in the A pipe, the centrifugal 10min of 500r/min removes large stretch of fragment tissue;

(3) supernatant liquor is transferred in the B pipe, centrifugal 2 times of 2600r/min is respectively 15 and 10min, removes the plastids such as chloroplast(id);

(4) get supernatant liquor to the C pipe, centrifugal 15 min of 10000 r/min abandon supernatant, add the A liquid of 1 mL precooling in precipitation, blow afloat gently precipitation, mixing with the rifle head.The centrifugal 10min of 1000r/min sucks supernatant in the D pipe, adds the 100mg/mL DNaseI of 1/500 times of volume and the 1mol/L MgCl of 1/100 times of volume ₂The 0.5mol/L Na that adds 1/25 times of volume behind the ice bath 1h ₂EDTA leaves standstill 10 min, stops the DNaseI reaction;

(5) the centrifugal 15min of 13200r/min removes supernatant, obtains the plastosome precipitation, adds ST liquid purifying 1 time again, obtains the plastosome of purifying;

(6) add the 1mL lysate B precipitation that suspends in the D pipe precipitation, add again 25mg/mL Proteinase K 2.5 μ L, 50 ℃ of incubation 1 h, 37 ℃ of incubation 1h again, during the interval shake;

(7) take out the centrifuge tube room temperature and place, after its cooling, add isopyknic phenol: chloroform: the primary isoamyl alcohol mixed solution, mixed solution mixed volume ratio is 25:24:1, fully mixing is placed on and shakes 15min on the shaking table, centrifugal 10 min of 10000 r/min;

(8) turn supernatant liquor to the E pipe, add isopyknic chloroform: the primary isoamyl alcohol mixed solution, mixed solution mixed volume ratio is 24:1, turns upside down to shake up approximately 10min centrifugal 10 min of 10000 r/min;

(9) supernatant liquor is transferred in the F pipe, then adds the RNaseT of the 1000U/ μ L of the RNaseA of 1/100 volume 10mg/mL and 1/500 volume ₁RNA is removed in 37 ℃ of water-baths 1 hour;

(10) add isopyknic chloroform: the primary isoamyl alcohol mixed solution, mixed solution mixed volume ratio 24:1, turning upside down shakes up approximately 10min, centrifugal 10 min of 10000 r/min;

(11) get 0. 1 times of volume NaAc and 2 times of volume dehydrated alcohols that supernatant liquor adds precooling to the centrifuge tube of 1.5mL ,-20 ℃ are spent the night;

(12) 4 ℃, the centrifugal 15min of 13200r/min abandon supernatant, and precipitation is with 75% washing with alcohol 2 ~ 3 times;

(13) dissolve in a small amount of water after being deposited in air drying ,-20 ℃ of refrigerations are for subsequent use.

Reagent

(1) buffer A: 1mol/L Tris-HCl 12.5mL, 0.5mol/L Na ₂EDTA 12.5mL, 1.3 mol/L NaCl 15.743g are settled to 250mL with the sterilization distilled water, autoclave sterilization, room temperature preservation.Add 0.5g BSA, 0.125g halfcystine, 750 μ L beta-mercaptoethanols before using.

(2) ST liquid: sucrose 13.6916g, 1mol/L Tris-HCl 5mL, 0.5mol/L Na ₂EDTA 5mL is settled to 100mL with the sterilization distilled water, autoclave sterilization, room temperature preservation.Add 0.1g BSA before using.

(3) lysate B:1mol/L Tris-HCl 2.5mL, 0.5mol/L Na ₂EDTA 4mL, 10% SDS 5mL is settled to 100mL with the sterilization distilled water, room temperature preservation.

As shown in Figure 2, can find out near the point sample hole have RNA to pollute without macromole DNA pollution and front end.Measure its OD with the nucleic acid-protein instrument ₂₆₀/ OD ₂₈₀And concentration, the result shows OD ₂₆₀/ OD ₂₈₀All between 1.8-2.0, show that mtDNA purity is higher, protein contamination is less.

Should be understood that, for those of ordinary skills, can be improved according to the above description or conversion, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.

Claims (1)

1. rapidly and efficiently extraction and the purification process of Corn Mitochondria DNA is characterized in that, may further comprise the steps:

(1) the corn explant is divided in the mortar that is filled to precooling, add the Extraction buffer A of precooling in the ratio of every gram sample 4mL, put into 1-2g quartz sand, be ground to homogenate; The centrifuge tube of getting 3 10mL is numbered respectively A, B, C, and the centrifuge tube of getting again 3 2mL is numbered respectively D, E, F;

(2) filter homogenate with 6 layers of sterile gauze, will grind tissue and put back in the mortar, add buffer A by every gram sample 2mL and again grind and filter, 2 times filtrate merges; To install to filtrate dividing in the A pipe, the centrifugal 10min of 500r/min removes large stretch of fragment tissue;

(3) supernatant liquor is transferred in the B pipe, centrifugal 2 times of 2600r/min is respectively 15 and 10min, removes the plastids such as chloroplast(id);

(4) get supernatant liquor to the C pipe, centrifugal 15 min of 10000 r/min abandon supernatant, add the A liquid of 1 mL precooling in precipitation, blow afloat gently precipitation, mixing with the rifle head; The centrifugal 10min of 1000r/min sucks supernatant in the D pipe, adds the 100mg/mL DNaseI of 1/500 times of volume and the 1mol/L MgCl of 1/100 times of volume ₂The 0.5mol/L Na that adds 1/25 times of volume behind the ice bath 1h ₂EDTA leaves standstill 10 min, stops the DNaseI reaction;

(5) the centrifugal 15min of 13200r/min removes supernatant, obtains the plastosome precipitation, adds ST liquid purifying 1 time again, obtains the plastosome of purifying;

(6) add the 1mL lysate B precipitation that suspends in the D pipe precipitation, add again 25mg/mL Proteinase K 2.5 μ L, 50 ℃ of incubation 1 h, 37 ℃ of incubation 1h again, during the interval shake;

(7) take out the centrifuge tube room temperature and place, after its cooling, add isopyknic phenol: chloroform: the primary isoamyl alcohol mixed solution, mixed solution mixed volume ratio is 25:24:1, fully mixing is placed on and shakes 15min on the shaking table, centrifugal 10 min of 10000 r/min;

(8) turn supernatant liquor to the E pipe, add isopyknic chloroform: the primary isoamyl alcohol mixed solution, mixed solution mixed volume ratio is 24:1, turns upside down to shake up approximately 10min centrifugal 10 min of 10000 r/min;

(9) supernatant liquor is transferred in the F pipe, then adds the RNaseT of the 1000U/ μ L of the RNaseA of 1/100 volume 10mg/mL and 1/500 volume ₁RNA is removed in 37 ℃ of water-baths 1 hour;

(10) add isopyknic chloroform: the primary isoamyl alcohol mixed solution, mixed solution mixed volume ratio 24:1, turning upside down shakes up approximately 10min, centrifugal 10 min of 10000 r/min;

(11) get 0. 1 times of volume NaAc and 2 times of volume dehydrated alcohols that supernatant liquor adds precooling to the centrifuge tube of 1.5mL ,-20 ℃ are spent the night;

(12) 4 ℃, the centrifugal 15min of 13200r/min abandon supernatant, and precipitation is with 75% washing with alcohol 2 ~ 3 times;

(13) dissolve in a small amount of water after being deposited in air drying ,-20 ℃ of refrigerations are for subsequent use;

Used reagent wherein:

(1) buffer A: 1mol/L Tris-HCl 12.5mL, 0.5mol/L Na ₂EDTA 12.5mL, 1.3 mol/L NaCl 15.743g are settled to 250mL with the sterilization distilled water, autoclave sterilization, room temperature preservation; Add 0.5g BSA, 0.125g halfcystine, 750 μ L beta-mercaptoethanols before using;

(2) ST liquid: sucrose 13.6916g, 1mol/L Tris-HCl 5mL, 0.5mol/L Na ₂EDTA 5mL is settled to 100mL with the sterilization distilled water, autoclave sterilization, room temperature preservation; Add 0.1g BSA before using;

(3) lysate B:1mol/L Tris-HCl 2.5mL, 0.5mol/L Na ₂EDTA 4mL, 10% SDS 5mL is settled to 100mL with the sterilization distilled water, room temperature preservation.

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