CN106957842B

CN106957842B - Extraction method of BAC clone DNA

Info

Publication number: CN106957842B
Application number: CN201710337946.0A
Authority: CN
Inventors: 张进平; 张晓春
Original assignee: Qingdao Andebei Life Technology Co ltd; Affiliated Hospital of University of Qingdao
Current assignee: Qingdao Andebei Life Technology Co ltd; Affiliated Hospital of University of Qingdao
Priority date: 2017-05-15
Filing date: 2017-05-15
Publication date: 2020-05-22
Anticipated expiration: 2037-05-15
Also published as: CN106957842A

Abstract

The invention relates to the fields of molecular biology, molecular genetics and cell biology, in particular to a method for extracting and purifying DNA. The invention adopts magnetic bead separation and molecular sieve filtration technology, combined with protease digestion technology, and invents a stable extraction method of BAC cloned DNA. The invention utilizes optimized suspension, lysis and neutralization buffer to obtain crude DNA precipitate; after resuspending the DNA precipitation, under suitable pH and ion concentration, the DNA polynucleotide macromolecules are nonspecifically and reversibly bound to Functional groups of magnetic nanomaterials; through rapid binding and cleaning steps, contaminants such as ions, proteins, carbohydrates and impurities such as small molecules and impurities such as small molecules in the cleavage process are removed; after further digestion, purification and filtration by protease, the purified product is produced. DNA product. The invention is simple and stable in operation, economical and efficient, non-toxic and non-polluting. The invention has excellent expansibility, and can realize standardized kit series products and fully automatic operation.

Description

Extraction method of BAC clone DNA

Technical Field

The invention relates to the fields of molecular biology, molecular genetics and cell biology, in particular to a method for extracting and purifying DNA.

Background

In recent decades, molecular biology and cytogenetics have been rapidly developed, and techniques related to sequencing, cloning, functional analysis and identification of DNA have been widely and deeply applied in the fields of medicine, agriculture and forestry, environment, oceans, forensic medicine, and the like. BAC (bacterial specific chromosome) cloning is the main method for storing and amplifying DNA large fragments, especially genomic DNA large fragments at present. BAC clones generally vary in length from 100kb to 300 kb. Due to the larger insert DNA of the BAC clone, the number of copies produced after transfection of E.coli was lower. Both the existing ion exchange column method and the manual extraction method have some defects and difficulties which are difficult to overcome.

Existing extraction andthe purification method still mainly adopts an ion exchange column separation method. Such as the plasmid maxi Kit (Cat No.12163) from QIAGEN, Omega Bio-tek

BAC/PAC DNA Maxi Kit (D2154-01), these commercial kits are generally able to meet the extraction requirements of less than 10ug and fragment sizes of less than 100kb in daily experiments. The yield of DNA harvested by ion exchange column extraction is generally low, and is sometimes undesirable, especially in large fragment DNA extraction processes. Also, large volume commercial extraction kits are generally expensive. Some kits also have certain reliability and stability problems.

In order to overcome the problem of column extraction and save cost, most of the research and application technicians adopt a manual operation method to extract DNA, but most of the methods use toxic and harmful organic solvents such as phenol, chloroform and the like, and the method has great harm to the experimenters and the environment. Meanwhile, the manual extraction process also easily causes the pollution of DNA products by organic solvents, and directly influences downstream experiments.

By adopting the traditional manual extraction, the degradation degree of DNA is high, and the amount of DNA products in 500ml of culture solution is generally far less than 10 ug. The small molecule fragments after DNA degradation also affect the accuracy of DNA metering. FIG. 1 shows the result of DNA precipitation using alkaline lysis method, followed by purification with a mixture of phenol-chloroform-isoamyl alcohol (25:24:1) and finally isopropanol (isopropyl alcohol). It is evident from FIG. 1 that there are a large number of small fragments of degraded DNA at positions less than 100 bp. These small molecule fragments cause the absorbance of the nucleic acid at 260nm to be amplified excessively, so that the absorbance at 260nm in the spectrophotometer exhibits an abnormally high value, resulting in a ratio of 260/280 to 260/230 that is much greater than normal. Such abnormal 260 readings can lead to errors in DNA metering.

Disclosure of Invention

The invention aims to overcome the problems of easy hydrolysis, small quantity, easy pollution and high cost of DNA in the extraction process of the large-fragment DNA, provides an extraction technology for extracting DNA with high quality, sufficient quantity, no toxicity, no pollution and low cost from a large-volume BAC clone bacteria culture solution, fundamentally solves the extraction and purification problems of the BAC clone large-fragment DNA, and simultaneously provides the possibility of automatically purifying the large-fragment DNA.

The invention is realized by adopting the following technical scheme:

a method for extracting BAC clone DNA mainly comprises the following steps:

(1) BAC clone E.coli culture: adding a micro amount of frozen stock solution of E.coli (Escherichia coli) BAC clone into 2mL of LB culture solution, performing initial culture for 4-6 hours, preparing a clone flat plate from the initial culture solution, picking out the clone from the flat plate cultured overnight, and implanting the clone into 500mL of LB culture solution containing corresponding antibiotics for overnight culture;

(2) bacterial lysis: centrifuging 500mL of escherichia coli culture solution to obtain bacterial precipitates, suspending, cracking, neutralizing and centrifuging the bacterial precipitates, removing most of cracked impurities, and extracting supernatant in the bacterial precipitates;

(3) DNA precipitation: mixing the supernatant containing the DNA with a certain volume of isopropanol and centrifuging to obtain a crude DNA precipitate;

(4) first extraction and cleaning: suspending the crude DNA precipitate by TE buffer solution, mixing the crude DNA precipitate with magnetic beads in a certain proportion, and cleaning the magnetic beads to obtain relatively pure DNA;

(5) and (3) secondary extraction and cleaning: purifying the DNA obtained in the step (4) by magnetic beads in a certain proportion;

(6) DNA repurification and concentration: and the DNA extracted by the magnetic beads is further concentrated by a 10kD ultrafiltration centrifugal tube to remove trace salt, low molecular weight nucleic acid and protein impurities, and finally a high-quality and high-purity DNA product is obtained.

In the above technical solution, further, before the second extraction and washing in step (5), the DNA obtained in step (4) is added to a protease digestion solution, and the residual protein is digested by an enzymatic reaction.

In the technical scheme, further, 500mL of escherichia coli culture solution is poured into two 500mL centrifuge tubes averagely in the step (2), and the escherichia coli culture solution is centrifuged for 10 minutes at 4 ℃ under the centrifugal force of 7000 Xg; adding 6mL of bacterial suspension buffer solution into the bacterial sediment, stirring the bacterial sediment on a high-speed oscillator by using a pipette, finally thoroughly scattering and suspending the bacterial sediment by using a pipette gun and a 10mL pipette, and transferring the scattered bacterial sediment into two 50mL centrifuge tubes; adding 6mL of bacterial lysate into a centrifuge tube, slightly inverting for 4-5 times, standing for 3-5 minutes at room temperature, immediately adding 6mL of neutralizing solution into the lysed thalli, slightly inverting and uniformly mixing for 4-5 times, placing on ice, after 3-5 minutes until the floccules of the thalli are completely turned from brown to white, placing the centrifuge tube on a centrifuge rotor, and centrifuging for 10 minutes at 12000Xg at 4 ℃.

In the above technical solution, further, the specific method for removing the cracked impurities in step (2) is as follows: placing the centrifuged centrifuge tube on ice, extracting supernatant in the centrifuge tube by using a pipette, gradually transferring all the supernatant into a filter tube, filtering the supernatant through cotton yarns at the bottom of the filter tube under the action of gravity, and naturally flowing into a 50mL centrifuge tube until all the supernatant is collected in the centrifuge tube; the manufacturing steps of the filter pipe are as follows: taking a 10mL syringe, shearing medical sterilized absorbent cotton yarn with the diameter of 5 square centimeters by using scissors, filling the medical sterilized absorbent cotton yarn at the bottom in the syringe by using a syringe plunger, and then pulling out the plunger; the filter tube is fixedly arranged in the 50mL centrifuge tube, and the nozzle faces the 50mL centrifuge tube.

In the above technical solution, further, the specific method for obtaining the crude DNA precipitate in step (3) is as follows: calculating the volume of the supernatant in the centrifuge tube, adding pure isopropanol into the centrifuge tube according to 0.7 time of the volume of the supernatant, arranging the centrifuge tube on a centrifugal rotor, centrifuging for 15 minutes at 4 ℃ at 4000Xg, centrifuging the DNA precipitate after centrifugation for 1 minute at 4000Xg after supernatant removal, removing the supernatant to obtain a precipitate after primary extraction of semitransparent DNA, immediately adding 500 mu LTE buffer solution into the DNA precipitate, and suspending and dissolving the precipitate in TE buffer solution by using a pipette gun.

In the technical scheme, further, the DNA suspension is transferred to 900 μ L (1.8X) Agencour XP magnetic beads in the step (4), and the DNA suspension is reversed, fully mixed and then kept stand for 5 minutes at room temperature to fully combine the DNA with the magnetic beads; placing the mixture on a magnetic frame after slight centrifugation, and standing for 3 to 5 minutes; discarding all supernatant with pipette or negative pressure, adding 1mL 70% alcohol, closing the centrifuge tube cover, rotating the centrifuge tube on a magnetic rack for 3-4 times to make the magnetic beads fully washed in alcohol, discarding alcohol, adding fresh 1mL 70% alcohol again, and repeating the washing process once; cleaning the magnetic beads for 2 times, discarding alcohol, centrifuging the magnetic beads, placing the magnetic beads on a magnetic rack again, and thoroughly discarding residual alcohol by using a pipette; standing at room temperature for 30 minutes, or heating in a heating plate at 37 deg.C for 5 minutes, standing in a magnetic frame until the magnetic bead mass begins to generate fine cracks due to drying; adding 200 mu L of TE buffer solution preheated at 65 ℃ into the dried magnetic beads, fully mixing uniformly, centrifuging, placing on a magnetic frame for 5 minutes, extracting supernatant, and transferring to a 1.5mL experiment tube, wherein the experiment tube contains eluted DNA; adding 200 μ L of TE buffer solution preheated at 65 deg.C again to extract magnetic beads, mixing well, centrifuging, placing on magnetic frame for 5 min, extracting supernatant, and mixing extracted DNA supernatant.

In the technical scheme, further, in the step (5), the DNA solution extracted for the first time is transferred to 650 μ L (1.6x) Agencour XP magnetic beads, and after being uniformly mixed, the mixture is kept stand at room temperature for 5 minutes, so that the DNA and the magnetic beads are fully combined; placing the mixture on a magnetic frame after slight centrifugation, and standing for 3 to 5 minutes; discarding all supernatant with a pipette or negative pressure, adding 1mL of 70% alcohol, closing the centrifuge tube cover, rotating the centrifuge tube on a magnetic rack for 3-4 times to fully wash the magnetic beads in the alcohol, discarding the alcohol, adding fresh 1mL of 70% alcohol again, and repeating the washing process once; cleaning the magnetic beads for 2 times, discarding alcohol, centrifuging the magnetic beads, placing the magnetic beads on a magnetic rack again, and thoroughly discarding residual alcohol by using a pipette; standing at room temperature for 30 minutes, or heating in a heating plate at 37 deg.C for 5 minutes, standing in a magnetic frame until the magnetic bead mass begins to generate fine cracks due to drying; adding 200 μ L of TE buffer solution preheated at 65 ℃ into the dried magnetic beads, fully mixing uniformly, centrifuging, placing on a magnetic frame for 5 minutes, extracting supernatant, and transferring to a 1.5mL experiment tube, wherein the experiment tube contains eluted DNA; adding 200 μ L of TE buffer solution preheated at 65 deg.C again to extract magnetic beads, mixing well, centrifuging, placing on magnetic frame for 5 min, extracting supernatant, and mixing extracted DNA supernatant.

In the technical scheme, further, the extracted DNA solution is transferred to a 0.5mL 10kD ultrafiltration centrifugal tube in the step (6), the DNA solution is centrifuged for 10 minutes at 14000Xg at 4 ℃, the filtrate is removed, TE buffer solution is added into the column core again to 400 mu L, the column core is centrifuged again for 10 minutes after being shaken up and down and mixed evenly, the volume scale of the centrifugal column core is observed, and the centrifugation is stopped until the liquid level is lower than 100 mu L; the core was inverted and centrifuged in a new collection tube and the DNA concentrate was collected by centrifugation at 14000Xg for 1 minute.

The process of BAC clone DNA cleavage and washing is a key step. The failure of DNA extraction is often due to lack of control in this process. In the process of the invention, after the bacterial suspension is added with the alkaline lysis solution, the lysis time is too long, the lysis reaction temperature is too high, the lysed DNA is quickly hydrolyzed, and a large amount of dragging bands or small molecule aggregation bands exist in the agarose gel. Therefore, the reaction time is shortened as much as possible in the step after the addition of the lysate. Finally, TE buffer solution with certain protection effect on DNA is used for completely replacing reagents applied in the extraction process or generated pollutants, thereby reducing the hydrolysis degree of DNA.

The invention adopts the magnetic bead separation and molecular sieve filtration technology, combines the protease digestion technology, and invents the stable extraction method of BAC clone DNA. The invention relates to key steps of an optimized extraction buffer solution system, magnetic bead combination, protease digestion, small molecule separation, concentration and the like; obtaining a crude DNA precipitate by using an optimized suspension, lysis and middle buffer solution and matching with a chaotropic agent and ions; through heavy suspension DNA precipitation, DNA polynucleotide macromolecules are non-specifically and reversibly combined to functional groups of the magnetic nano material, such as carboxyl and silicon, under the appropriate pH and ion concentration, and pollutants such as ions, proteins, saccharides and the like and impurities such as small molecules and the like in the cracking process are removed through rapid combination and washing steps.

The method can effectively extract DNA with fragments more than 10kb, and the treated volume is usually 100mL to 500mL of conventional bacterial culture solution; high salt, protein, sugar and small molecular impurities can be effectively removed from the large-volume culture solution, the stability and the product quality of the DNA purification process are improved to a great extent, and the large degradation of the DNA in the extraction process is effectively overcome; the extracted DNA has high quality and sufficient quantity, and 50-100 micrograms of high-purity DNA can be stably obtained in every 500mL of culture solution; toxic and harmful organic solvents such as phenol, chloroform and the like are not adopted, so that the method is friendly to the experimenters and the environment, and organic solvent pollution to DNA products is avoided; the operation is simple and stable, and the method is economical and efficient; the kit has excellent expansibility, and can realize standardized kit series products and full-automatic operation; fundamentally solves the problem of extraction and purification of BAC clone large-fragment DNA which needs to be solved urgently and is involved in DNA analysis, in particular to genomic DNA analysis experiment, and has great significance for meeting the requirements of DNA molecular probe marking and industry thereof on the large-fragment DNA.

Drawings

FIG. 1 is a diagram of agarose gel electrophoresis of DNA extracted by alkaline lysis;

FIG. 2 is a schematic structural diagram of a simple filter device according to an embodiment;

wherein, 1, a filter pipe; 2. centrifuging the tube; 3. absorbent cotton yarn;

FIG. 3 is a diagram showing the state of a DNA precipitate obtained after filtering the lysis impurities according to the example;

FIG. 4 is a diagram showing a state in which a fine crack is formed in a lump of magnetic beads at the start of drying;

FIG. 5 is an agarose gel electrophoresis of BAC DNA extracted by the method of example.

Detailed Description

In order to make the above objects, features and advantages of the present invention more clearly understood, the present invention will be further described with reference to the accompanying drawings and examples.

Examples

1. Main equipment and reagent material

A floor type centrifuge: thermosphere RC 6+ centrifuge and rotor F12-6x500, F13-14x50cy

Bench top 4 ℃ centrifuge: eppendorf 5424R

Conventional pocket centrifuge

37 ℃ incubator

Shaking table for culturing bacteria at 37 DEG C

50mL centrifuge tube: thermo Scientific, 339653

1.5ml of the tube, without DNase (deoxyribonuclease) and RNase (ribonuclease).

And (3) ultra-filtration centrifuge tube: merck Millipore, Amicon Ultra 0.5mL Centrif μ ge Filters, Ultracel 10K

LB bacterial culture solution: the following reagents were added to 800mL of deionized purified water, 10g of Bacto-tryptone, 5g of yeast extract, 10g of NaCl, pH adjusted to 7.5 with NaOH, and then deionized purified water was added to 1 liter. Sterilizing at high temperature for later use.

Antibiotic working solution: and (4) diluting the antibiotic powder to a required concentration according to the requirement of BAC clone on corresponding antibiotics. If chloramphenicol is generally used for BAC cloning, a quantitative amount of powdered chloramphenicol antibiotic can be diluted to 50mg/mL with 100% ethanol and added to a final concentration of 12.5. mu.g/mL in a conventional culture solution.

Bacterial suspension buffer (TE buffer): 6.06g Tris and 3.72g Na2EDTA.2H2O were dissolved in 800mL deionized purified water, the pH was adjusted to 8.0 with HCl, and then water was added to 1 liter. 100mg of RNase A was added to a final concentration of 100. mu.g/mL. The suspension buffer after RNase A addition was stored at 4 ℃.

Bacterial lysate: 8.0g of NaOH pellets were dissolved in 900mL of deionized purified water, and 50mL of 20% SDS (w/v) was added, and 1000mL of water was added.

Neutralizing liquid: 294.5g of Potasssium Acetate was dissolved in 500mL of deionized purified water, then the pH was adjusted to 5.5 with about 110mL of glacial acetic acid, and finally water was added to 1 liter.

Nucleic acid purification kit: agencourt AMPure XP, 60mL, A63881, Beckman Coulter

Protease K solution: (20mg/mL) x5, RNA grade,25530049, Thermo Fisher

Coli culture of BAC clones

(1) Preparing a monoclonal selection flat plate: adding 2g of agarose powder into a 250mL disinfection bottle, adding 100mLLB culture solution, and uniformly mixing; after sterilizing at 210 ℃ for 20 minutes under high temperature and high pressure, cooling to 50 ℃, and then quickly adding chloramphenicol with the final concentration of 12.5 ug/mL. After mixing, the mixture was quickly poured into about 9 10mL culture plates, each plate was poured into about 10mL, completely cooled at room temperature, and stored in a 4 ℃ freezer for further use.

(2) 2mL of LB medium was added to a 10mL round bottom bacterial culture tube and the corresponding chloramphenicol was added to a final concentration of 12.5 ug/mL.

(3) BAC clones frozen at-80 ℃ were transferred onto dry ice and kept frozen all the time, and a small amount of the frozen stock solution was dipped with a sterilized 200. mu.L or 1mL pipette tip and transferred to 2mL of LB medium.

(4) The tube to which the BAC clone E.coli had been added was transferred to a shaker at 37 ℃ per 250 rpm for about 6-8 hours and cultured to a slightly turbid transparent state.

(5) The BAC clone culture solution is diluted by LB culture solution according to the proportion of 1 to 10000. For example, 10. mu.L of the culture medium is added to 990. mu.L of LB medium, mixed well, and then 10. mu.L of the mixture is added to 990. mu.L of fresh LB medium.

(6) The inoculation toothpick or the inoculation loop is inserted into the diluted clone culture solution, and then the three bisection areas of the culture flat plate are sequentially and lightly scribed by the inoculation loop. One plate was inoculated at a time.

(7) The inoculated plates were inverted and incubated overnight in an incubator at 37 ℃ for 20 hours until monoclonal colonies were formed. The flat plate after the formation of the monoclonal colony is sealed by parafilm or preservative film, and the flat plate is stored in a refrigerator at 4 ℃. The storage and use period is generally not more than 5 days.

(8) Adding 500mL of LB culture solution into a triangular culture flask with the volume of 1000mL, adding chloramphenicol into the culture solution to a final concentration of 12.5ug/mL, and mixing the mixture after gentle shaking. Single colony was picked from BAC clone plate and inoculated into LB culture solution in a triangular culture flask. The triangular culture bottle mouth is sealed by tin foil paper. After shaking gently, the mixture was fixed on a shaker at 37 ℃ and 260 rpm for cultivation. The bacteria were cultured to a confluent state of about 80% in pale color. The incubation time is usually not more than 20 hours.

3. Bacterial lysis and DNA precipitation

500mL of bacterial culture incubated for about 20 hours were poured on average into two 500mL centrifuge tubes, equilibrated and placed in a centrifuge rotor (F12-6X500LEX) and centrifuged at 7000Xg for 10 minutes at 4 ℃ in a centrifuge (Sorvall RC 6+, Thermo Scientific).

After centrifugation, the supernatant was discarded, and the precipitated cells were retained. The pellet can also be stored with the centrifuge tube at-20 ℃ until use.

And (3) respectively adding 6mL of bacterial suspension buffer solution into the centrifuged thalli, stirring the thalli sediment on a high-speed oscillator by using a pipette, and finally thoroughly scattering and suspending the thalli by using a pipette gun and a 10mL pipette. The disrupted cells were transferred to two 50mL (Thermofisiher, 339653) centrifuge tubes.

Add 6mL of bacterial lysate to the centrifuge tube, invert gently 4 to 5 times, and stand no more than 5 minutes at room temperature.

Immediately adding 6mL of neutralizing solution into the lysed thalli, gently inverting and uniformly mixing for 4 to 5 times, placing on ice, and after about 3 to 5 minutes, completely converting the thalli floccules from brown to white.

The tubes were mounted in a centrifuge rotor (F13-14X50cy) and centrifuged at 12000Xg for 10 minutes at 4 ℃.

A simple filtration device was made with a 10mL syringe and a 50mL centrifuge tube at the centrifugation gap. A small piece of sterilized absorbent cotton yarn of about 5 cm square was cut with scissors and packed in the bottom of the syringe by a syringe plunger, and then the plunger was pulled off, and the filter tube assembly was attached to a 50mL centrifuge tube with adhesive tape, with the nozzle of the syringe facing the inside of the centrifuge tube, as shown in FIG. 2.

The centrifuged tubes were carefully placed on ice and the supernatant from 2 tubes was carefully removed with a 10mL pipette, taking care to avoid taking the flocculent precipitate. And transferring all the supernatants into a filter tube one by one, filtering the supernatants through cotton yarns at the bottom of the syringe under the action of gravity, naturally flowing into a 50mL centrifuge tube until all the supernatants are collected in the centrifuge tube, and discarding the filter device. Most of impurities seen by naked eyes after cracking can be removed after the device is used for filtering.

The volume of the supernatant in the centrifuge tube was calculated and pure isopropanol was added to the centrifuge tube at 0.7 times the volume of the supernatant. For example, if the total volume of the filtered supernatant is 25mL, 17.5mL of 100% isopropanol should be added, and the final isopropanol content is 41% and the final total volume is 42.5 mL.

The tube was mounted on a centrifuge rotor (F13-14X50cy) and centrifuged at 4000Xg for 15 minutes at 4 ℃.

After centrifugation, a 2mL low binding centrifuge tube was charged with 900. mu.L of AgencourtXP magnetic beads mixed by vortexing, and the tube was gently centrifuged by a tabletop centrifuge and then placed at room temperature for use. Should be ready at least 10 minutes prior to use.

After the centrifuged DNA pellet was carefully discarded, the supernatant was centrifuged at 4000Xg for 1 minute, and the supernatant was discarded as much as possible by a pipette. At this time, the sidewall of the bottom of the centrifuge tube has a translucent precipitate after primary extraction of DNA, as shown in FIG. 3, and the arrow indicates the precipitate after primary extraction of DNA. Particular attention must be paid at this time to the fact that the centrifuge tube cannot be left at room temperature for a long time to prevent degradation of DNA.

4. First extraction and cleaning

Immediately, 500. mu.L of TE buffer was added to the DNA pellet and the pellet was suspended and dissolved in TE buffer using 1000. mu.L pipette.

mu.L of the DNA suspension was transferred to 900. mu.L (1.8X) of Agencour XP beads which had been prepared at room temperature, inverted and mixed well and then allowed to stand at room temperature for 5 minutes to allow the DNA to bind well to the beads.

The mixture was centrifuged in a conventional mini-centrifuge for 2 seconds, placed in a magnetic rack (DynaMagTM-2Magnet, ThermoFisher,12321D), and allowed to stand for 3 to 5 minutes.

Removing all supernatant with pipette or negative pressure, adding 1mL of 70% ethanol, closing the centrifuge tube cover, and rotating the centrifuge tube 3-4 times on the magnetic frame to make the magnetic beads fully washed in the ethanol. Finally, the alcohol was discarded, and a fresh 1mL 70% alcohol was added again and the washing process was repeated once.

Washing the magnetic beads for 2 times, discarding alcohol, centrifuging the magnetic beads, placing the magnetic beads on a magnetic rack again, and completely discarding residual alcohol by using a pipette. Standing at room temperature for more than 30 minutes, or heating in a heating plate at 37 deg.C for 5 minutes and standing in a magnetic frame until the magnetic bead mass begins to generate fine cracks due to drying, as shown in FIG. 4.

To the dried beads, 200. mu.L of a TE buffer solution preheated at 65 ℃ was added, well mixed, centrifuged, and placed in a magnetic stand for 5 minutes, and the supernatant was carefully extracted and transferred to a 1.5mL tube containing the eluted DNA. Adding 200 μ L of TE buffer solution preheated at 65 deg.C again to extract magnetic beads, mixing well, centrifuging, placing on magnetic frame for 5 min, and collecting supernatant. The extracted DNA supernatants were combined in a total volume of 400. mu.L.

5. Protease digestion

This step may be an option. Since there may still be some histone, enzyme, sugar and so on residue in the DNA extract, for the following experiment, further protease digestion treatment can be performed to remove the residual protein.

10. mu.L of 20mg/mL protease K solution was added to 400. mu.L of the extracted DNA extract, mixed well and then incubated in a 37 ℃ water bath for 4 hours.

6. Second extraction and cleaning

650. mu.L (1.6X) of Agencourt XP magnetic beads were added to a 2mL low binding centrifuge tube and allowed to stand at room temperature for use.

Transferring the DNA solution extracted for the first time or the DNA extract treated by the protease K into 650 mu L of magnetic beads, uniformly mixing, and standing for 5 minutes at room temperature.

And (4) slightly centrifuging the magnetic beads after standing, placing the magnetic beads on a magnetic frame, and standing for 3 to 5 minutes.

The supernatant was discarded by pipette or negative pressure, 1mL of 70% ethanol was added, the centrifuge tube lid was closed, and the centrifuge tube was rotated 3 to 4 times on the magnetic stand to wash the magnetic beads sufficiently in ethanol. The alcohol was discarded and a fresh 1mL 70% alcohol was added again and the washing process was repeated once.

Washing the magnetic beads for 2 times, discarding alcohol, centrifuging the magnetic beads, placing the magnetic beads on a magnetic rack again, and completely discarding residual alcohol by using a pipette. Standing at room temperature for more than 30 min, or heating in a heating plate at 37 deg.C for 5 min, standing in magnetic frame until the magnetic bead mass shows fine cracks due to drying.

To the dried beads, 200. mu.L of a TE buffer preheated at 65 ℃ was added, mixed well, centrifuged, and placed in a magnetic stand for 5 minutes, and the supernatant was carefully extracted and transferred to a 1.5ml tube containing the eluted DNA. Adding 200 μ L of TE buffer solution preheated at 65 deg.C again to extract the magnetic beads, mixing well, centrifuging, placing on a magnetic frame for 5 min, and collecting the supernatant. The extracted DNA supernatants were pooled at about 400. mu.L.

Repurification and concentration of DNA

The extracted DNA solution was transferred to Amicon Ultra 0.5mL of a LCenterifugal filter (10kD, UFC501024) manufactured by Millipore corporation, and centrifuged at 14000Xg in a 4 ℃ bench centrifuge for 10 minutes according to the manufacturer's request. The filtrate was removed, TE buffer was added again to the column core to 400. mu.L, and the mixture was gently shaken up and down and then centrifuged again for 10 minutes. The volume scale of the core of the column was observed and centrifugation was stopped until the liquid level was below 100. mu.L.

The core was inverted and centrifuged in a new collection tube and the DNA concentrate was collected by centrifugation at 14000Xg for 1 minute.

8. Quality inspection

Nanodrop2000C measures DNA product quantity (quality) and quality (quality). mu.L of DNA solution is added to the Nanodrop2000C assay platform, and the ratio of 260nm to 280nm of the obtained DNA is between 1.80 and 1.90. Nucleic acid small molecule contaminants may still be present at ratios above 1.90. The ratio of 260nm to 230nm should be equal to or slightly higher than 2.0, and if higher than 2.2, it indicates that there is still contamination of the salt molecules.

Agarose gels were used to observe the Integrity of the DNA (Integrity). 0.4 g of agar was added to a heated flask pre-filled with 40ml of TAE buffer, mixed well and heated in a microwave oven for about 1 minute to dissolve well, taking care not to boil out of the vial. Adding about 5 μ l of 10mg/mL ethidium bromide solution, shaking gently, mixing, pouring into agar gel preparation device, removing bubbles on the surface of the gel, inserting into a cell comb, and cooling at room temperature for 30 min to solidify completely. mu.L of the DNA solution was mixed with the loading solution and loaded, and the positive and negative control samples and the appropriate DNA ladder were added at the same time. The DNA results were examined under 365nm UV light after running the gel at about 100v for about 30 minutes at about 5 v/cm.

Lanes

2 and 3 shown in FIG. 5 show the result of BAC DNA, which should be at a position of 10kb or more.

The invention can effectively extract DNA with high quality, sufficient quantity, no toxicity, no pollution and low cost from the large-volume BAC clone bacteria culture solution; the method has the advantages of simple and stable operation, economy, high efficiency, no toxicity and no pollution. The invention has excellent expansibility, and can realize standardized kit set series products and full-automatic operation.

The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.

Claims

1. a method for extracting BAC clone DNA, is characterized in that, its main steps comprise:

(1) BAC clone Escherichia coli culture: Add a trace amount of E.coli (Escherichia coli) BAC clone's cryopreserved stock solution to 2 mL of LB culture solution and carry out initial culture for 4 to 6 hours, and make a clone plate from the initial culture solution. Pick clones from the overnight culture plate and plant them in 500 mL of LB medium containing the corresponding antibiotics for overnight culture;

(2) Bacterial lysis: centrifuge 500 mL of E. coli culture solution to obtain bacterial sediment, which is then suspended, lysed, neutralized, and centrifuged to remove most of the lysed impurities, and extract the supernatant;

(3) DNA precipitation: mix the DNA-containing supernatant with isopropanol 0.7 times the volume of the supernatant and centrifuge to obtain a crude DNA precipitate;

(4) The first extraction and cleaning: the crude DNA precipitate is suspended in TE buffer and mixed with magnetic beads, and the magnetic beads are washed to obtain relatively pure DNA;

(5) Second extraction and cleaning: the DNA obtained in step (4) is purified by magnetic beads again;

(6) DNA repurification and concentration: DNA extracted by magnetic beads is further concentrated by 10kD ultrafiltration centrifuge tube to remove trace salt, low molecular weight nucleic acid and protein impurities, and finally obtain high quality and high purity DNA product.

2. the extraction method of BAC clone DNA according to claim 1, is characterized in that, before carrying out step (5) extraction for the second time, cleaning, the DNA that step (4) obtains is added protease digestion solution, through enzyme The reaction digests residual protein.

3. the extraction method of BAC clone DNA according to claim 1 and 2, is characterized in that, described step (2) 500mL Escherichia coli culture liquid is poured into two 500ml centrifuge tubes on average, 4 ℃ under 7000×g centrifugal force Centrifuge for 10 minutes; add 6ml of bacterial suspension buffer to the bacterial sediment, stir up the bacterial sediment with a pipette on a high-speed shaker, and finally use a pipette and a 10ml pipette to completely disperse the bacterial cells. Suspend, transfer the disintegrated cells to two 50ml centrifuge tubes; add 6ml of bacterial lysate to the centrifuge tubes, invert gently 4 to 5 times, let stand for 3 to 5 minutes at room temperature, and immediately add lysate to the centrifuge tube. Add 6ml of neutralizing solution to the cells in the bacterium, gently invert and mix 4 to 5 times, and place on ice. After 3 to 5 minutes, the flocs of the cells are completely changed from brown to white. Place the centrifuge tube in The rotor was centrifuged and centrifuged at 12000 xg for 10 minutes at 4°C.

4. the extraction method of BAC clone DNA according to claim 3, is characterized in that, the concrete method that described step (2) removes the impurity after cracking is: centrifuge tube after centrifugation is placed on ice, with pipetting Extract the supernatant in the centrifuge tube, transfer all the supernatant to the filter tube one by one, and filter the supernatant through the cotton yarn at the bottom of the filter tube under the action of gravity, and then flow naturally into the 50mL centrifuge tube until all the supernatant is completely Collected in a centrifuge tube; the manufacturing steps of the filter tube are: take a 10ml syringe, cut out 5 square centimeters of medical sterilized absorbent cotton yarn with scissors, and use a syringe to push the plug to fill the inner bottom of the syringe, and then pull out the push plug; The filter tube is fixedly arranged in the 50mL centrifuge tube, and the spout faces the 50mL centrifuge tube.

5. the extraction method of BAC clone DNA according to claim 4, is characterized in that, the concrete method that described step (3) obtains DNA crude sediment is: calculate the supernatant volume in centrifuge tube, according to supernatant 0.7 times the volume of the liquid was added to the centrifuge tube with pure isopropanol, placed in the centrifuge rotor, and centrifuged at 4000×g for 15 minutes at 4°C. minutes, discard the supernatant to obtain a translucent precipitate after preliminary DNA extraction, immediately add 500 μl of TE buffer to the DNA precipitate, and use a pipette to suspend and dissolve the precipitate in the TE buffer.

6. The method for extracting BAC clone DNA according to claim 5, wherein in the step (4), the DNA suspension is transferred to 900 μl (1.8×) Agencourt XP magnetic beads, and after inversion and adequate mixing Let stand for 5 minutes at room temperature to fully bind the DNA to the magnetic beads; centrifuge in a pocket benchtop centrifuge for 2 seconds, place it on a magnetic stand, and let it stand for 3 to 5 minutes; discard all supernatant with a pipette or negative pressure, Add 1ml of 70% alcohol, close the cap of the centrifuge tube, spin the centrifuge tube 3 to 4 times on the magnetic stand, discard the alcohol, add fresh 1ml of 70% alcohol again, and repeat the washing process; after washing the magnetic beads twice, Discard the alcohol, centrifuge the magnetic beads and place them on the magnetic stand again, and use a pipette to completely discard the residual alcohol; let stand for 30 minutes at room temperature, or warm them up in a 37°C heating plate for 5 minutes and then stand on the magnetic stand , until the magnetic beads clumps began to appear small cracks due to drying; add 200 μl of TE buffer preheated at 65 °C to the dried magnetic beads, mix well, centrifuge and place on a magnetic rack for 5 minutes, extract the supernatant, transfer To a 1.5ml experimental tube, which contains the eluted DNA; add 200μl of 65°C pre-warmed TE buffer again to extract the magnetic beads, mix well, centrifuge and place on a magnetic stand for 5 minutes, extract the supernatant, and combine the extracted DNA supernatant.

7. the extraction method of BAC clone DNA according to claim 6, is characterized in that, described step (5) transfers the DNA solution extracted for the first time to 650 μl (1.6x) Agencourt XP magnetic beads, after mixing Let stand at room temperature for 5 minutes, gently centrifuge and place on a magnetic stand for 3 to 5 minutes; use a pipette or negative pressure to discard all supernatant, add 1ml of 70% alcohol, close the cap of the centrifuge tube, Spin the centrifuge tube 3 to 4 times on the magnetic stand, discard the alcohol, add fresh 1ml 70% alcohol again, and repeat the washing process; after washing the magnetic beads twice, discard the alcohol, centrifuge the magnetic beads and place them on the magnetic Remove the residual alcohol completely with a pipette; let it stand for 30 minutes at room temperature, or warm it up in a 37°C heating plate for 5 minutes and then place it on a magnetic stand, until the magnetic bead clumps begin to appear small due to drying. Cracks; add 200 μl of TE buffer preheated at 65°C to the dried magnetic beads, mix well, centrifuge and place on a magnetic stand for 5 minutes, extract the supernatant and transfer it to a 1.5 ml experimental tube, which contains the eluted DNA; Add 200 μl of pre-warmed TE buffer at 65°C again to extract the magnetic beads, mix well, centrifuge and place on a magnetic stand for 5 minutes, extract the supernatant, and combine the extracted DNA supernatant.

8. the extraction method of BAC clone DNA according to claim 7, is characterized in that, described step (6) transfers the extracted DNA solution in 0.5mL 10kD ultrafiltration centrifuge tube, at 4 ℃ with 14000×g centrifugation 10 minutes , remove the filtrate, add TE buffer to the column again to 400μl, shake up and down to mix, and then centrifuge again for 10 minutes, observe the volume scale of the centrifuge column, stop centrifugation until the liquid level is lower than 100μl; invert; Centrifuge the inner core in a new collection tube and centrifuge at 14,000 x g for 1 minute to collect the DNA concentrate.