CN106957842A - BAC clones DNA extracting method - Google Patents

Abstract

The present invention relates to molecular biology and molecular genetics and cell biology, DNA extraction and purification process is related specifically to.Present invention employs Beads enrichment and molecular sieve filtration technology, associated proteins enzymic digestion technology has invented the stable extracting method that BAC clones DNA.The present invention obtains rough DNA precipitations using the suspension, cracking, neutralization buffer of optimization；Precipitated through resuspended DNA, under suitable pH and ion concentration, by DNA polynucleotides macromolecular non-specificity and the functional group for being reversibly bound to magnetic Nano material；By quickly combining, cleaning step, remove the impurity such as pollutant and small molecule such as ion, albumen, carbohydrate of cracking process；Further digested through protease, purify and the concentration of filter after produce the DNA product of purifying.Present invention stabilization simple to operate, economical and efficient, nontoxic pollution-free.The present invention possesses excellent autgmentability, can realize the kit series product and full-automatic operation of standardization.

Description

Extraction method of BAC cloned 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 technique

In the past ten years, molecular biology and cytogenetics have developed rapidly, and technologies involving DNA sequencing, cloning, functional analysis and identification have been widely and deeply applied in fields such as medicine, agriculture and forestry, environment, oceanography, and forensic science. BAC (bacterial artificial chromosome) cloning is currently the main method for storing and amplifying large DNA fragments, especially large genomic DNA fragments. The length of BAC clones generally ranges from 100kb to 300kb. Due to the large insert DNA fragment of the BAC clone, the copy number produced after transfection into E. coli is low. Both the existing ion-exchange column method and the manual method of extraction have some defects and difficulties that are difficult to overcome.

The existing extraction and purification methods still mainly adopt ion exchange column separation method. Such as QIAGEN's PlasmidMaxi Kit (CatNo.12163), Omega Bio-tek's BAC/PAC DNA Maxi Kit (D2154-01), these commercial kits can generally meet the extraction needs of less than 10ug and fragment size less than 100kb in daily experiments. The yield of DNA harvested by the ion exchange column extraction method is generally low, especially in the process of extracting large fragments of DNA, sometimes it is not ideal. Likewise, large-capacity commercial extraction kits are generally more expensive. Some kits also have reliability and stability issues to some extent.

In order to overcome the problem of column extraction and to save costs, many research and application technicians use manual methods to extract DNA, but most of them use toxic and harmful organic solvents such as phenol and chloroform, which are harmful to experimenters and The environmental hazards are great. At the same time, the manual extraction process can easily lead to contamination of DNA products by organic solvents, which directly affects downstream experiments.

Using traditional manual extraction, the degree of degradation of DNA is high, and the amount of DNA product from 500ml culture medium is generally much lower than 10ug. Small molecule fragments after DNA degradation can also affect the accuracy of DNA metering. Figure 1 shows the results of the DNA obtained by applying the alkaline lysis method, followed by purification with a mixture of phenol-chloroform-isoamyl alcohol (25:24:1), and finally precipitated with isopropyl alcohol. It can be clearly observed from Figure 1 that there are a large number of small molecular fragments after DNA degradation at positions below 100 bp. These small molecular fragments will cause the abnormal amplification of the absorbance value of nucleic acid at 260nm, so that the absorbance value of the spectrophotometer at 260nm will show an abnormally high value, making the ratio of 260/280 and 260/230 much larger than the normal amount. . This abnormal 260 reading can lead to errors in DNA metering.

Contents of the invention

The purpose of the present invention is to overcome the problems of easy hydrolysis, small quantity, easy pollution and high cost of DNA existing in the above-mentioned large fragment DNA extraction process, and provide a method of extracting high-quality, sufficient quantity and non-toxic DNA from the large-volume BAC clone bacterial culture solution. , pollution-free, low-cost DNA extraction technology fundamentally solves the problem of extraction and purification of large fragments of DNA from BAC clones, and at the same time provides the possibility of automatic purification of large fragments of DNA.

The present invention is realized by adopting the following technical solutions:

A method for extracting BAC clone DNA, the main steps of which include:

(1) BAC clone Escherichia coli culture: Add a small amount of frozen stock solution of E.coli (Escherichia coli) BAC clone 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 500mL LB medium containing corresponding antibiotics for overnight culture;

(2) Bacterial lysing: Centrifuge 500mL of E. coli culture solution to obtain bacterial precipitates, which are then suspended, lysed, neutralized, and centrifuged to remove most of the lysed impurities and extract the supernatant;

(3) DNA precipitation: the DNA-containing supernatant was mixed with a certain volume of isopropanol and centrifuged to obtain a crude DNA precipitate;

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

(5) The second extraction and cleaning: the DNA obtained in step (4) is purified by a certain proportion of magnetic beads;

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

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

The above technical scheme, further, in the step (2) pour 500mL E. coli culture solution into two 500ml centrifuge tubes on average, and centrifuge at 7000xg centrifugal force for 10 minutes at 4°C; add 6mL bacterial suspension buffer to the bacterial sediment , use a pipette on a high-speed oscillator to disperse the bacterial sediment, and finally use a pipette gun and a 10mL pipette to completely disperse and suspend the bacterial cells, and transfer the dispersed bacterial cells to two 50mL centrifuge tubes Add 6mL of bacterial lysate to the centrifuge tube, invert gently 4 to 5 times, let stand at room temperature for 3 to 5 minutes, immediately add 6mL of neutralizing solution to the lysed bacteria, invert gently for 4 5 times, place on ice, and after 3 to 5 minutes until the bacterial flocs completely turn from brown to white, place the centrifuge tube in a centrifuge rotor and centrifuge at 12000xg for 10 minutes at 4°C.

In the above technical scheme, further, the specific method for removing the lysed impurities in the step (2) is: place the centrifuged tube on ice, extract the supernatant in the centrifuged tube with a pipette, and put all the above The supernatant is transferred to the filter tube successively, and the supernatant flows naturally into the 50mL centrifuge tube after being filtered by the cotton yarn at the bottom of the filter tube under the action of gravity, until all the supernatant is collected in the centrifuge tube; the manufacturing steps of the filter tube It is: take a 10mL syringe, cut out 5 square centimeters of medical sterilized absorbent cotton yarn with scissors, and use the syringe to push the plug to fill it in the bottom of the syringe, and then pull out the push plug; the filter tube is fixed in the 50mL centrifuge tube, and the spout faces the 50mL Centrifuge tube.

The above technical scheme, further, the specific method for obtaining the crude DNA precipitate in the step (3) is: calculate the volume of the supernatant in the centrifuge tube, and add pure isopropyl isopropyl to the centrifuge tube according to 0.7 times the volume of the supernatant Alcohol, place the centrifuge tube in the centrifuge rotor, centrifuge at 4000xg for 15 minutes at 4°C, discard the supernatant after centrifuging the DNA pellet, then centrifuge at 4000xg for 1 minute, discard the supernatant, and obtain translucent DNA For the precipitate after preliminary extraction, immediately add 500 μL TE buffer to the DNA precipitate and suspend and dissolve the precipitate in TE buffer with a pipette gun.

The above technical scheme, further, in the step (4), transfer the DNA suspension to 900 μL (1.8x) Agencourt XP magnetic beads, invert and mix well and let stand at room temperature for 5 minutes, so that the DNA and magnetic beads Fully combine; place on the magnetic stand after gentle centrifugation, and let it stand for 3 to 5 minutes; discard all supernatant with a pipette gun or negative pressure, add 1mL of 70% alcohol, close the centrifuge tube cover, and put it on the magnetic stand Rotate the centrifuge tube 3 to 4 times to wash the magnetic beads in alcohol, discard the alcohol, add 1 mL of fresh 70% alcohol again, and repeat the washing process; after washing the magnetic beads twice, discard the alcohol, and centrifuge the magnetic beads Put it on the magnetic stand again, and use a pipette gun to completely discard the residual alcohol; let it stand at room temperature for 30 minutes, or heat it on a heating plate at 37°C for 5 minutes and then put it on the magnetic stand until the magnetic bead clumps begin to dry out Small cracks appear; add 200 μL 65°C preheated TE buffer to the dried magnetic beads, mix well, centrifuge and place on a magnetic stand for 5 minutes, extract the supernatant, transfer to a 1.5mL experimental tube, which contains washing Proposed DNA: add 200 μL 65°C preheated TE buffer to extract the magnetic beads again, mix well, centrifuge and place on the magnetic stand for 5 minutes, extract the supernatant, and combine the extracted DNA supernatant.

The above technical scheme, further, in the step (5), transfer the DNA solution extracted for the first time to 650 μL (1.6x) Agencourt XP magnetic beads, mix well and let stand at room temperature for 5 minutes, so that the DNA and the magnetic beads are fully mixed. Combine; gently centrifuge and place on a magnetic stand, let stand for 3 to 5 minutes; discard all supernatant with a pipette gun or negative pressure, add 1mL of 70% alcohol, close the cap of the centrifuge tube, and spin on the magnetic stand Centrifuge the tube 3 to 4 times to wash the magnetic beads in alcohol, discard the alcohol, add fresh 1mL 70% alcohol again, and repeat the washing process; after washing the magnetic beads twice, discard the alcohol, and centrifuge the beads Put it on the magnetic stand again, and use a pipette gun to completely discard the residual alcohol; let it stand at room temperature for 30 minutes, or heat it on a heating plate at 37°C for 5 minutes and then put it on the magnetic stand until the magnetic bead clumps begin to dry out Small cracks appear; add 200 μL of 65°C preheated TE buffer to the dried magnetic beads, mix well, centrifuge and place on a magnetic stand for 5 minutes, extract the supernatant, transfer to a 1.5mL test tube, which contains the eluted Add 200 μL 65°C preheated TE buffer to extract the magnetic beads again, mix well, centrifuge and place on the magnetic stand for 5 minutes, extract the supernatant, and combine the extracted DNA supernatant.

In the above technical scheme, further, in the step (6), transfer the extracted DNA solution to a 0.5mL 10kD ultrafiltration centrifuge tube, centrifuge at 14000xg for 10 minutes at 4°C, remove the filtrate, and add TE to the column core again Buffer solution to 400 μL, shake up and down to mix well, then centrifuge again for 10 minutes, observe the volume scale of the centrifugal column core, stop centrifuging until the liquid level is lower than 100 μL; invert the centrifuge core into a new collection tube, and centrifuge at 14000xg The DNA concentrate was collected for 1 min.

The lysing and cleaning process of BAC cloned DNA is a key link. The failure of DNA extraction is often due to the failure to master this link. During the invention, it was found that after adding the alkaline lysate to the bacterial suspension, the lysing time was too long, the cleavage reaction temperature was too high, and the lysed DNA was rapidly hydrolyzed, which was reflected in the agarose gel, and there were a large number of drag or small molecules gathering belt. Therefore, the reaction time should be kept as short as possible in the steps following the addition of the lysate. Finally, the TE buffer that has a certain protective effect on DNA should be used to completely replace the reagents used in the extraction process or the pollutants generated, thereby reducing the degree of DNA hydrolysis.

The invention adopts magnetic bead separation and molecular sieve filtration technology, combined with protease digestion technology, and invents a stable extraction method of BAC clone DNA. The present invention involves key steps such as optimized extraction buffer system, magnetic bead binding, protease digestion, small molecule separation, and concentration; using optimized suspension, lysis, and medium buffer, with chaotropic agents and ions to obtain crude DNA precipitation ; DNA polynucleotide macromolecules are non-specifically and reversibly bound to functional groups of magnetic nanomaterials, such as carboxyl and silicon groups, under suitable pH and ion concentration after resuspended DNA precipitation, through rapid combination, The cleaning step removes impurities such as ions, proteins, sugars and other pollutants and small molecules in the cracking process.

The method of the present invention can effectively extract the DNA whose fragment is larger than 10kb, and the volume to be processed is usually a conventional 100ml to 500mL bacterial culture solution; high salt, protein, sugar and small molecular impurities can be effectively removed from the large-volume culture solution. It greatly improves the stability and product quality of the DNA purification process, and effectively overcomes the large amount of DNA degradation during the extraction process; the extracted DNA is of high quality and sufficient quantity, and 50 to 100 micrograms of high-purity DNA can be stably obtained per 500 mL of culture medium; Does not use toxic and harmful organic solvents such as phenol and chloroform, is friendly to experimenters and the environment, and will not cause organic solvent pollution to DNA products; simple and stable operation, cost-effective; has excellent scalability and can realize a standardized kit set series products and fully automated operation; it fundamentally solves the problem of extracting and purifying large fragments of DNA from BAC clones that involves DNA analysis, especially genomic DNA analysis experiments. The need for large fragments of DNA makes perfect sense.

Description of drawings

Fig. 1 is the DNA agarose gel electrophoresis figure that is extracted for application alkaline lysis method;

Fig. 2 is the structural representation of embodiment simple filter device;

Among them, 1. filter tube; 2. centrifuge tube; 3. absorbent cotton yarn;

Fig. 3 is the state diagram of the DNA precipitate obtained after filtering and cleaving impurities in the embodiment;

Fig. 4 is the state diagram when the magnetic bead agglomerate starts to appear tiny cracks due to drying;

Fig. 5 is an agarose gel electrophoresis image of BAC DNA extracted by the method of the embodiment.

detailed description

In order to understand the above-mentioned purpose, features and advantages of the present invention more clearly, the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Example

1. Main equipment and reagent materials

Floor type centrifuge: ThermoSorval RC 6+ centrifuge and rotor F12-6x500, F13-14x50cy

Benchtop 4°C centrifuge: Eppendorf 5424R

Conventional pocket centrifuge

37℃ incubator

37°C Bacterial Culture Shaker

50mL centrifuge tube: Thermo Scientific, 339653

1.5mL Eppendorf centrifuge tube, free of DNase (deoxyribonuclease) and RNase (ribonuclease).

Ultrafiltration centrifuge tube: Merck Millipore, Amicon Ultra 0.5mL Centrif μge Filters, Ultracel 10K

LB bacterial culture solution: add the following reagents to 800mL deionized pure water, 10g Bacto-tryptone, 5gyeast extract, 10g NaCl, adjust the pH to 7.5 with NaOH, then add deionized pure water to 1 liter. High temperature disinfection for use.

Antibiotic working solution: Dilute the antibiotic powder to the required concentration according to the corresponding antibiotic requirements of BAC clone. If chloramphenicol is generally used in BAC clones, a certain amount of powdered chloramphenicol antibiotic can be weighed and diluted to 50 mg/mL with 100% alcohol, and added to conventional culture medium to a final concentration of 12.5 μg/ml.

Bacterial Suspension Buffer (TE Buffer): Dissolve 6.06g Tris and 3.72g Na2EDTA·2H2O in 800mL deionized pure water, adjust the pH to 8.0 with HCL, then add water to 1 liter. Add 100 mg RNase A to a final concentration of 100 μg/mL. The suspension buffer after adding RNase A should be stored at 4°C.

Bacterial Lysis Solution: Dissolve 8.0g NaOH particles in 900mL deionized pure water, finally add 50mL 20% SDS (w/v), and finally add water to 1000mL.

Neutralizing solution: Dissolve 294.5g of Potassium Acetate in 500mL of deionized pure water, then adjust the pH to 5.5 with about 110mL of glacial acetic acid, and finally add water to 1 liter.

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

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

2. BAC clone E. coli culture

(1) Monoclonal selection plate preparation: Add 2g of agarose powder to a 250mL sterilized bottle, then add 100mL of LB culture solution, mix well; sterilize at 210°C for 20 minutes and cool to 50°C, then quickly add the final concentration of 12.5ug/mL of chloramphenicol. After mixing evenly on the ultra-clean workbench, quickly pour it into about nine 10mL culture plates, pour about 10mL into each plate, cool completely at room temperature and store in a 4°C refrigerator for later use.

(2) Add 2 mL of LB culture medium to a 10 mL round-bottom bacterial culture tube, and add the corresponding chloramphenicol antibiotic to a final concentration of 12.5 ug/ml.

(3) Transfer the BAC clones frozen at -80°C to dry ice and keep them in a frozen state. Use a sterilized 200 μL or 1 mL pipette tip to dip a small amount of freezing solution and transfer it to 2 mL of LB culture medium.

(4) Move the culture tube in which BAC cloned E. coli has been added to a shaker at 37°C/250 rpm for about 6-8 hours, until it is slightly turbid and transparent.

(5) Dilute the BAC clone culture solution with LB culture solution at a ratio of 1:10000. For example, first take 10 μL of the culture solution and add it to 990 μL of the LB culture solution, mix well, then take 10 μL of it and add it to 990 μL of fresh LB culture solution.

(6) Insert the inoculation toothpick or inoculation loop into the diluted cloning culture solution, and then use the inoculation loop to lightly draw lines in three equally divided areas of the culture plate in sequence. Inoculate one plate at a time.

(7) Invert the inoculated plate and incubate overnight in a 37°C incubator for 20 hours until monoclonal colonies form. After the formation of monoclonal colonies, the flat plate should be sealed with parafilm or plastic wrap, and the flat plate should be stored upside down in a refrigerator at 4°C. The storage and use period is generally not more than 5 days.

(8) Add 500mL LB culture medium to a 1000mL Erlenmeyer flask, add chloramphenicol at a final concentration of 12.5ug/mL to the culture medium, shake gently and mix well. Single clonal colonies were picked from the BAC cloning plate and inoculated into the LB medium in the Erlenmeyer flask. Seal the mouth of the triangle culture bottle with tin foil. Shake gently and fix in a culture shaker at 37°C, 260 rpm. Bacteria were grown to about 80% confluency in pale white color. The incubation time usually does not exceed 20 hours.

3. Bacterial Lysis and DNA Precipitation

Pour 500mL of bacterial culture cultured for about 20 hours into two 500mL centrifuge tubes, balance and place in a centrifugal rotor (F12-6x500LEX), and centrifuge at 7000xg in a centrifuge (Sorvall RC 6+, Thermo Scientific) at 4°C 10 minutes.

After centrifugation, discard the supernatant and keep the precipitated cells. At this time, the precipitate can also be stored at -20°C together with the centrifuge tube for future use.

Add 6mL of bacterial suspension buffer to the centrifuged bacteria respectively, and use a pipette to stir up the bacteria sediment on a high-speed oscillator, and finally use a pipette gun and a 10mL pipette to completely break up and suspend the bacteria. Transfer the broken up cells to two 50mL (Thermofisher, 339653) centrifuge tubes.

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

Immediately add 6mL of neutralizing solution to the lysed cells, gently invert and mix 4 to 5 times, place on ice, and wait for about 3 to 5 minutes until the cell flocs completely turn from brown to white.

Place the centrifuge tube in a centrifuge rotor (F13-14x50cy), and centrifuge at 12000xg for 10 minutes at 4°C.

In the centrifugation gap, use a 10mL syringe and a 50mL centrifuge tube to make a simple filter device. Use scissors to cut out a small piece of medical sterilized absorbent cotton yarn about 5 square centimeters, and use a syringe to push the plug to fill it in the inner bottom of the syringe, then pull out the plug, and use adhesive tape to paste the filter tube device made in the 50mL centrifuge tube, pay attention to the syringe The spout faces into the centrifuge tube, as shown in Figure 2.

Carefully place the centrifuged tubes on ice, and carefully extract the supernatant from the two centrifuged tubes with a 10mL pipette, taking care not to get flocculent precipitates as much as possible. Transfer all the supernatant to the filter tube one by one, let the supernatant pass through the cotton gauze filter at the bottom of the syringe under the action of gravity, and then flow naturally into the 50mL centrifuge tube until all the supernatant is collected in the centrifuge tube, discard the filter device . After being filtered by the device, most of the impurities visible to the naked eye after cracking can be removed.

Calculate the volume of the supernatant in the centrifuge tube, and add pure isopropanol to the centrifuge tube according to 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, the final isopropanol content is 41%, and the final total volume is 42.5mL.

Place the centrifuge tube in a centrifuge rotor (F13-14x50cy), and centrifuge at 4000xg for 15 minutes at 4°C.

During centrifugation, add 900 μL AgencourtXP magnetic beads vortexed to a 2mL low binding centrifuge tube, centrifuge gently in a desktop centrifuge, and place at room temperature for later use. It should be prepared at least 10 minutes before use.

After the centrifuged DNA pellet was carefully discarded, the supernatant was discarded, and then centrifuged at 4000xg for 1 minute, and the supernatant was discarded as much as possible with a pipette. At this time, there is a translucent precipitate after the preliminary extraction of DNA on the side wall of the bottom of the centrifuge tube, as shown in Figure 3, the arrow points to the precipitate after the preliminary extraction of DNA. At this time, special attention should be paid not to leave the centrifuge tube at room temperature for a long time to avoid DNA degradation.

4. The first extraction and cleaning

Immediately add 500 μL of TE buffer to the DNA pellet and suspend and dissolve the pellet in TE buffer with a 1000 μL pipette.

Transfer 500 μL of the DNA suspension to 900 μL (1.8x) Agencourt XP magnetic beads that have been prepared at room temperature, invert and mix well, then let stand at room temperature for 5 minutes to fully bind the DNA to the magnetic beads.

Centrifuge for 2 seconds with a conventional pocket centrifuge, then place on a magnetic stand (DynaMagTM-2Magnet, ThermoFisher, 12321D), and let stand for 3 to 5 minutes.

Use a pipette gun or negative pressure to discard all the supernatant, add 1mL of 70% alcohol, close the cap of the centrifuge tube, and rotate the centrifuge tube 3 to 4 times on the magnetic stand to fully wash the magnetic beads in the alcohol. Finally discard the alcohol, add fresh 1mL 70% alcohol again, and repeat the cleaning process.

After washing the magnetic beads twice, discard the alcohol, centrifuge the magnetic beads and place them on the magnetic stand again, and completely discard the residual alcohol with a pipette gun. Let it stand at room temperature for more than 30 minutes, or heat it on a heating plate at 37°C for 5 minutes and then place it on the magnetic stand until the magnetic bead clumps begin to appear small cracks due to drying, as shown in Figure 4.

Add 200 μL of 65°C preheated TE buffer to the dried magnetic beads, mix well, centrifuge and place on a magnetic stand for 5 minutes, carefully extract the supernatant, and transfer it to a 1.5mL experimental tube, which contains the eluted DNA. Add 200 μL of 65°C preheated TE buffer again to extract the magnetic beads, mix thoroughly, centrifuge, place on the magnetic stand for 5 minutes, and extract the supernatant. Combine the extracted DNA supernatants to a total volume of 400 µL.

5. Protease Digestion

This step is optional. Since there may still be a certain amount of histones, enzymes, sugars and other residues in the DNA extraction solution, further protease digestion can be performed to remove residual proteins for subsequent experiments.

Add 10 μL of Proteinase K solution with a concentration of 20 mg/mL to the extracted 400 μL of DNA extract, mix well and incubate in a 37°C water bath for 4 hours.

6. The second extraction and cleaning

Add 650 μL (1.6x) Agencourt XP magnetic beads to a 2mL low binding centrifuge tube and let stand at room temperature for later use.

Transfer the DNA solution extracted for the first time or the DNA extraction solution treated with Proteinase K to 650 μL magnetic beads, mix well and let stand at room temperature for 5 minutes.

Gently centrifuge the static beads and place them on a magnetic stand for 3 to 5 minutes.

Discard the supernatant with a pipette or negative pressure, add 1 mL of 70% alcohol, close the cap of the centrifuge tube, and rotate the centrifuge tube 3 to 4 times on the magnetic stand to fully wash the magnetic beads in the alcohol. Discard the alcohol, add fresh 1mL 70% alcohol again, and repeat the cleaning process once.

After washing the magnetic beads twice, discard the alcohol, centrifuge the magnetic beads and place them on the magnetic stand again, and completely discard the residual alcohol with a pipette gun. Let it stand at room temperature for more than 30 minutes, or heat it on a 37°C heating plate for 5 minutes and then place it on a magnetic stand until the magnetic bead clumps show fine cracks due to drying.

Add 200 μL of 65°C preheated TE buffer to the dried magnetic beads, mix well, centrifuge and place on a magnetic stand for 5 minutes, carefully extract the supernatant, and transfer it to a 1.5ml experimental tube, which contains the eluted DNA. Add 200 μL 65°C preheated TE buffer again to extract the magnetic beads, mix well, centrifuge and place on the magnetic stand for 5 minutes, and extract the supernatant. Combine the extracted DNA supernatant to about 400 μL.

7. DNA repurification and concentration

The extracted DNA solution was transferred to Amicon Ultra 0.5mL centrifugal filter (10kD, UFC501024) produced by Millipore Company, and centrifuged at 14000xg for 10 minutes in a desktop centrifuge at 4°C according to the manufacturer's requirements. Remove the filtrate, add TE buffer to the column core again to 400 μL, shake it up and down to mix well, and centrifuge again for 10 minutes. Observe the volume scale of the spin cartridge until the liquid level falls below 100 μL and stop centrifuging.

Invert the centrifuge core in a new collection tube and centrifuge at 14,000xg for 1 minute to collect the DNA concentrate.

8. Quality Check

Nanodrop2000C measures DNA product quantity (quantity) and quality (quality). Take 1.5 μL of DNA solution and add it to the Nanodrop2000C assay platform, and the ratio of 260nm to 280nm of the obtained DNA should be between 1.80 and 1.90. If the ratio is higher than 1.90, there may still be small nucleic acid molecule pollutants. The ratio of 260nm to 230nm should be equal to or slightly higher than 2.0. If it is higher than 2.2, it indicates that there are still pollutants of salt molecules.

Agarose gel was used to observe the integrity of DNA (Integrity). Add 0.4 g of agar to a heating bottle pre-filled with 40 mL of TAE buffer, mix well and heat in a microwave oven for about 1 minute until fully dissolved, being careful not to boil out of the bottle. Add about 5 μl of ethidium bromide solution with a concentration of 10mg/mL, shake gently and mix well, then quickly pour into the agar gel preparation device, remove the air bubbles on the surface of the gel, insert the well comb, and cool at room temperature for 30 minutes until it is completely solidified. Take 1 μL of DNA solution and mix it with the sample solution before loading the sample. At the same time, add the positive and negative control samples and the appropriate DNA ladder (ladder) when loading the sample. Run the gel at 100v for about 30 minutes according to 5v/cm, and check the DNA results under 365nm ultraviolet light. Lanes 2 and 3 shown in Figure 5 are the results of BAC DNA, which should be above 10kb.

The invention can effectively extract high-quality, sufficient quantity, non-toxic, pollution-free and low-cost DNA from large-volume BAC cloning bacterial culture solution; the invention is simple and stable in operation, economical and efficient, non-toxic and pollution-free. The invention has excellent expansibility, and can realize a series of standardized kits and sets of products and fully automatic operation.

The above-mentioned embodiments are only descriptions of preferred implementations of the present invention, and are not intended to limit the scope of the present invention. Variations and improvements should fall within the scope of protection defined by the claims of the present invention.

Claims (8)

1. a kind of BAC clones DNA extracting method, it is characterised in that its key step includes：

(1) BAC clone E. colis culture：Micro Escherichia coli BAC clones are added in 2mL LB nutrient solutions freezes stoste And carry out initial incubation 4 to 6 hours, clone square position, the picked clones from the square position of incubated overnight are made from initial incubation liquid And it is implanted into incubated overnight in LB nutrient solutions of the 500mL containing corresponding antibiotic；

(2) bacteria lysis：500mL E. coli broth is centrifuged, bacterial precipitation thing is obtained, bacterial precipitation passes through again Suspend, crack, neutralize, centrifugation removes the impurity after most of cracking, extracts wherein supernatant；

(3) DNA is precipitated：Supernatant containing DNA is mixed and centrifuged with the isopropanol of certain volume, the rough precipitations of DNA are obtained Thing；

(4) extracting for the first time, cleaning：The rough sediments of DNA are mixed after being suspended through TE buffer solutions with a certain proportion of magnetic bead, are cleaned Magnetic bead, is obtained compared with pure dna；

(5) second of extracting, cleaning：The DNA that step (4) is obtained is again through a certain proportion of magnetic beads for purifying；

(6) DNA repuritys and concentration：The DNA extracted through magnetic bead is further concentrated by 10kD ultra-filtration centrifuge tubes, is removed micro Salt, low molecular weight nucleic acid and protein impurities, finally obtain high-quality high-purity DNA product.

2. BAC according to claim 1 clones DNA extracting method, it is characterised in that carrying out step (5) second Before extracting, cleaning, the DNA that step (4) is obtained adds protease digestion liquid, and remaining protein is digested through enzyme reaction.

3. BAC according to claim 1 or 2 clones DNA extracting method, it is characterised in that the step (2) will 500mL E. coli broths are averagely poured into two 500mL centrifuge tubes, and 4 DEG C centrifuge 10 minutes under 7000xg centrifugal force；To 6mL bacterial suspension buffer solutions are separately added into bacterial precipitation thing, bacterial sediment are stirred scattered with pipette on high-speed oscillator, most Thoroughly thalline is broken up with liquid-transfering gun and 10mL pipettes afterwards, suspended, the thalline after breaing up is transferred to two 50ml centrifuge tubes In；6ml bacterial lysates are separately added into centrifuge tube, are gently inverted 4 to 5 times, in being stored at room temperature 3 to 5 minutes, immediately to splitting Thalline in solution is separately added into 6ml neutralizers, and gently reversion is mixed 4 to 5 times, is positioned on ice, is wadded a quilt with cotton after 3 to 5 minutes to thalline Shape thing switchs to white by brown completely, and centrifuge tube is placed in into centrifugal rotor, is centrifuged 10 minutes with 12000xg in 4 DEG C.

4. BAC according to claim 3 clones DNA extracting method, it is characterised in that the step (2) removes cracking The specific method of impurity afterwards is：Centrifuge tube after centrifugation is placed on ice, the supernatant in centrifuge tube is extracted with pipette, will All supernatants are gradually transferred in screen pipe, and supernatant is natural after the cotton yarn filtering for filtering bottom of the tube under gravity Flow in 50mL centrifuge tubes, until all supernatants are all collected in centrifuge tube；The making step of the screen pipe is：Take 10ml Syringe, 5 square centimeters of medical sterilization degreasing cotton yarn is cut with scissors, and is clogged with syringe plunger in bottom in syringe Portion, then pulls out plunger；The screen pipe is fixedly installed in 50mL centrifuge tubes, and spout is towards 50mL centrifuge tubes.

5. BAC according to claim 4 clones DNA extracting method, it is characterised in that it is thick that the step (3) obtains DNA The specific method of sediment processed is：The supernatant volume in centrifuge tube is calculated, according to 0.7 times of supernatant volume into centrifuge tube Pure isopropanol is added, centrifuge tube is placed in centrifugal rotor, is centrifuged 15 minutes with 4000xg in 4 DEG C, the DNA after centrifugation is precipitated After abandoning supernatant, then with 4000xg centrifugations 1 minute, abandoning supernatant obtains the precipitation after translucent DNA is tentatively extracted Thing, 500 μ L TE buffer solutions are added into DNA sediments and sediment suspension is dissolved in TE buffer solutions with liquid-transfering gun immediately.

6. BAC according to claim 5 clones DNA extracting method, it is characterised in that the step (4) suspends DNA Liquid is transferred in 900 μ L (1.8x) Agencourt XP magnetic beads, is inverted and is fully mixed after standing 5 minutes at room temperature, makes DNA Fully combined with magnetic bead；Pocket desk-top centrifuge is positioned over magnetic frame after 2 seconds, stand 3 to 5 minutes；With liquid-transfering gun or negative Pressure discards all supernatants, adds 1mL 70% alcohol, closes centrifugation lid, and 3 to 4 centrifuge tubes are rotated on magnetic frame, Alcohol is discarded, the fresh alcohol of 1mL 70% is added again, cleaning process is repeated once；After magnetic bead is cleaned 2 times, alcohol is discarded, Magnetic frame is placed in after magnetic bead is centrifuged again, remaining alcohol is thoroughly discarded with liquid-transfering gun；30 minutes are stood at room temperature, or in Heating is statically placed in magnetic frame in 5 minutes again in 37 DEG C of heating dish, until magnetic bead agglomerate because drying starts to show tiny crack；To Dried magnetic bead adds the TE buffer solutions of 200 μ L, 65 DEG C of preheatings, is fully placed in magnetic frame 5 minutes after mixing, centrifugation, extracts Supernatant, is transferred to 1.5ml experiment tubes, wherein containing the DNA eluted out；The TE buffer solutions for adding 200 μ L, 65 DEG C of preheatings again are taken out Magnetic bead is carried, fully magnetic frame is placed in 5 minutes after mixing, centrifugation, extracts supernatant, merge the DNA supernatants extracted.

7. BAC according to claim 6 clones DNA extracting method, it is characterised in that the step (5) will for the first time The DNA solution of extraction is gone in 650 μ L (1.6x) Agencourt XP magnetic beads, is mixed after being stored at room temperature 5 minutes, is gently centrifuged After be positioned over magnetic frame, stand 3 to 5 minutes；All supernatants are discarded with liquid-transfering gun or negative pressure, 1mL70% alcohol is added, closed Upper centrifugation lid, 3 to 4 centrifuge tubes are rotated on magnetic frame, alcohol is discarded, the fresh alcohol of 1mL 70% is added again, weight A multiple cleaning process；After magnetic bead is cleaned 2 times, alcohol is discarded, magnetic frame is placed in after magnetic bead is centrifuged again, it is thorough with liquid-transfering gun Bottom discards remaining alcohol；30 minutes are stood at room temperature, or heating is statically placed in magnetic frame in 5 minutes again in 37 DEG C of heating dish, directly To magnetic bead agglomerate because drying starts to show tiny crack；The TE bufferings of 200 μ L, 65 DEG C of preheatings are added to dried magnetic bead Liquid, fully mixes, is placed in magnetic frame 5 minutes after centrifugation, extract supernatant, be transferred to 1.5mL experiment tubes, wherein containing eluting out DNA；The TE buffer extractions magnetic beads of 200 μ L, 65 DEG C of preheatings are added again, are fully placed in magnetic frame 5 minutes after mixing, centrifugation, Supernatant is extracted, merges the DNA supernatants extracted.

8. BAC according to claim 7 clones DNA extracting method, it is characterised in that the step (6) is by extraction DNA solution is transferred in 0.5mL 10kD ultra-filtration centrifuge tubes, is centrifuged 10 minutes with 14000xg in 4 DEG C, is removed filtered solution, again TE buffer solutions are added in Xiang Zhuxin to 400 μ L, are centrifuged again 10 minutes after jog mixing up and down, the volume of observation centrifugal column core is carved Degree, centrifugation is stopped until liquid level is less than after 100 μ L；Reversing centrifugal inner core is in a new collecting pipe, 14000xg centrifugations 1 Minute collects DNA concentrates.