CN106834274B - method for recovering fragmented degraded DNA - Google Patents
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Abstract
The invention discloses a method for recovering fragmented degraded DNA. The method comprises the following steps: adding carboxyl magnetic beads into a solution for degrading DNA, so that large-fragment DNA is combined with the carboxyl magnetic beads, and small-fragment DNA is kept in the solution; separating the carboxyl magnetic beads combined with the large fragment DNA from the solution containing the small fragment DNA; separating the large-fragment DNA from the carboxyl magnetic beads, and then fragmenting the large-fragment DNA until small-fragment DNA is formed; recovering small fragment DNA in the solution containing small fragment DNA and small fragment DNA formed by fragmenting large fragment DNA. The method for recovering the fragmented degraded DNA greatly improves the purification recovery rate of the fragmented degraded DNA, provides effective guarantee for the application of the sample in downstream molecular biology research, and has the advantages of simple reagent components, convenient operation, low cost, no need of special equipment and improvement of the recovery rate of the fragmented degraded DNA.
Description
Technical Field
the invention relates to a method for recovering fragmented degraded DNA, belonging to the technical field of molecular biology.
Background
DNA is the material basis of human genetics, and in order to clarify the functions and structures of DNA in human genetics and development processes, the detection of DNA sequences becomes the most important means for studying DNA. With the huge demand for DNA information, the second generation sequencing was born, which is based on a sequencing-by-synthesis technology, and represented by Roche (454), Illumina (Solexa), ABI (SOLID). The most basic technology of the second-generation sequencing technology is the construction of a second-generation sequencing library, wherein the library construction is started by firstly crushing and fragmenting the genome DNA of a sample to be tested, and the main means comprise shearing force, ultrasonic waves, nitrogen, enzyme digestion and the like.
the most critical factor affecting DNA fragmentation is the integrity of genomic DNA, and DNA with high integrity will achieve better results. However, most clinical samples are paraffin-embedded samples, and in recent years, many puncture and laser micro-cutting samples appear, which have small DNA content and high degradation degree. When the conventional DNA fragmentation and recovery means is used for processing the samples, the degradation DNA is fragmented and the fragments are too small to cause great loss in the purification process, so that the low fragmentation recovery rate of the degradation DNA becomes a bottleneck problem in the research of the samples by scientific researchers. How to improve the fragmentation recovery rate of degraded DNA and ensure the uniformity of DNA fragments as much as possible becomes a problem to be solved urgently.
disclosure of Invention
The invention mainly aims to provide a method for recovering fragmented degraded DNA, so as to overcome the defects in the prior art.
in order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
The embodiment of the invention provides a method for recovering fragmented degraded DNA, which comprises the following steps:
adding carboxyl magnetic beads into a solution for degrading DNA, so that large-fragment DNA is combined with the carboxyl magnetic beads, and small-fragment DNA is kept in the solution, wherein the size of the large-fragment DNA is larger than 2000bp, and the size of the small-fragment DNA is 200 bp-2000 bp;
separating the carboxyl magnetic beads combined with the large fragment DNA from the solution containing the small fragment DNA;
Separating the large-fragment DNA from the carboxyl magnetic beads, and then fragmenting the large-fragment DNA until small-fragment DNA is formed;
Recovering small fragment DNA in the solution containing small fragment DNA and small fragment DNA formed by fragmenting large fragment DNA.
In some embodiments, the method of recovering fragmented degraded DNA comprises the steps of:
(1) Dissolving degraded DNA in water to form a degraded DNA solution;
(2) adding carboxyl magnetic beads into the degraded DNA solution, uniformly mixing, and incubating at room temperature to fully combine the carboxyl magnetic beads with the large-fragment DNA;
(3) settling the carboxyl magnetic beads by using the action of a magnetic field until a mixed solution is clarified, separating the carboxyl magnetic beads, and collecting a clarified solution containing small-fragment DNA;
(4) washing the carboxyl magnetic beads;
(5) Drying the carboxyl magnetic beads at room temperature, eluting with eluent, and standing at room temperature;
(6) Clarifying the elution mixed solution obtained in the step (5) by using the action of a magnetic field, and separating out the clarified solution;
(7) Transferring the clear solution separated in the step (6) into a fragmenting instrument for fragmentation treatment, so that large fragment DNA is broken into small fragment DNA;
(8) combining the solution containing small fragment DNA obtained in the step (7) with the clarified solution containing small fragment DNA obtained in the step (3);
(9) separating the small fragment DNA from the solution containing the small fragment DNA finally obtained in the step (8).
in some embodiments, the concentration of degraded DNA in the solution of degraded DNA is between 1ug/30uL and 1ug/150 uL.
In some embodiments, the carboxyl magnetic beads are added to the solution for degrading DNA, and step (2) comprises: adding the carboxyl magnetic beads into the solution for degrading DNA, uniformly mixing, and incubating at room temperature for 5-20 min to ensure that the carboxyl magnetic beads are fully combined with the large-fragment DNA.
Wherein the carboxyl magnetic beads are 5mg/mL, and the dosage is 20uL-100 uL.
In some embodiments, step (4) comprises: and washing the carboxyl magnetic beads by using an ethanol solution with the concentration of 80 v/v%.
in some embodiments, step (5) comprises: drying the carboxyl magnetic beads at room temperature for 1min-10min, and then adding eluent for elution.
In some embodiments, the carboxyl magnetic beads bound to large fragments of degraded DNA are placed in a Covaris fragmentation instrument for fragmentation to small fragments of degraded DNA.
In some embodiments, the method for recovering fragmented degraded DNA is characterized in that step (9) comprises:
Uniformly mixing the solution containing the small fragment DNA finally obtained in the step (8) with a phosphate buffer solution, and adding the mixture into a centrifugal column;
Adding a PE buffer solution into the centrifugal column, and cleaning the centrifugal column;
Placing the empty centrifugal column in a centrifuge, centrifuging for 2min at full speed, and air drying at room temperature;
adding eluent into the centrifugal column to elute the small fragment DNA.
The eluent is Tris buffer salt solution with the concentration of 3mM-15mM, and the pH value is 7.3-8.5.
In some embodiments, the agent I is formulated by: 3mM-15mM Tris buffer is dissolved in ultrapure water, and then the pH value of the solution is adjusted to 7.3-8.5 by HCl solution.
In some embodiments, after washing treatment in the carboxyl magnetic beads combined with the large fragments of degraded DNA, drying is performed at room temperature for 1min to 10min, and then 30uL to 80uL of reagent I is added.
in some embodiments, after all of the small fragment DNA is separated from the spin column, a volume of reagent I ranging from 15uL to 80uL is added to the spin column, and the spin column is eluted at 20 ℃ to 70 ℃.
In some embodiments, the elution temperature employed in step (9) is from 20 ℃ to 70 ℃.
In some embodiments, the amount of elution buffer used in step (9) is 100 uL.
In some embodiments, the buffer comprises buffer PB (qiagen, germany) in an amount of 0.5mL to 3 mL.
Compared with the prior art, the invention has the advantages that: the method for recovering the fragmented degraded DNA greatly improves the purification recovery rate of the fragmented degraded DNA, designs the fragmented recovery method aiming at the degraded DNA compared with the existing DNA fragmentation recovery method, provides effective guarantee for the sample to be used for downstream molecular biology research, and has the advantages of simple reagent components, convenient operation, low cost, no need of special equipment and capability of improving the recovery rate of the fragmented degraded DNA.
Drawings
FIG. 1 is a graph showing a comparison of the amounts of DNA used in examples 1 to 4 of the present invention and in comparative examples.
FIG. 2 is a graph showing the comparison of the DNA recovery rates in examples 1 to 4 of the present invention and in the comparative example.
Detailed Description
In view of the deficiencies in the prior art, the inventors of the present invention have made extensive studies and extensive practices to provide technical solutions of the present invention. The technical solution, its implementation and principles, etc. will be further explained as follows.
the embodiment of the invention provides a method for recovering fragmented degraded DNA, which comprises the following steps:
adding carboxyl magnetic beads into a solution for degrading DNA, so that large-fragment DNA is combined with the carboxyl magnetic beads, and small-fragment DNA is kept in the solution, wherein the size of the large-fragment DNA is larger than 2000bp, and the size of the small-fragment DNA is 200 bp-2000 bp;
Separating the carboxyl magnetic beads combined with the large fragment DNA from the solution containing the small fragment DNA;
separating the large-fragment DNA from the carboxyl magnetic beads, and then fragmenting the large-fragment DNA until small-fragment DNA is formed;
recovering small fragment DNA in the solution containing small fragment DNA and small fragment DNA formed by fragmenting large fragment DNA.
in some embodiments, the method of recovering fragmented degraded DNA comprises the steps of:
(1) Dissolving degraded DNA in water to form a degraded DNA solution;
(2) adding carboxyl magnetic beads into the degraded DNA solution, uniformly mixing, and incubating at room temperature to fully combine the carboxyl magnetic beads with the large-fragment DNA;
(3) Settling the carboxyl magnetic beads by using the action of a magnetic field until a mixed solution is clarified, separating the carboxyl magnetic beads, and collecting a clarified solution containing small-fragment DNA;
(4) washing the carboxyl magnetic beads;
(5) Drying the carboxyl magnetic beads at room temperature, eluting with eluent, and standing at room temperature;
(6) clarifying the elution mixed solution obtained in the step (5) by using the action of a magnetic field, and separating out the clarified solution;
(7) Transferring the clear solution separated in the step (6) into a fragmenting instrument for fragmentation treatment, so that large fragment DNA is broken into small fragment DNA;
(8) Combining the solution containing small fragment DNA obtained in the step (7) with the clarified solution containing small fragment DNA obtained in the step (3);
(9) separating the small fragment DNA from the solution containing the small fragment DNA finally obtained in the step (8).
In some embodiments, the concentration of degraded DNA in the solution of degraded DNA is between 1ug/30uL and 1ug/150 uL.
In some embodiments, the carboxyl magnetic beads are added to the solution for degrading DNA, and step (2) comprises: adding the carboxyl magnetic beads into the solution for degrading DNA, uniformly mixing, and incubating at room temperature for 5-20 min to ensure that the carboxyl magnetic beads are fully combined with the large-fragment DNA.
wherein the using amount of the carboxyl magnetic beads is 20uL-100 uL.
In some embodiments, step (4) comprises: and washing the carboxyl magnetic beads by using an ethanol solution with the concentration of 80 v/v%.
In some embodiments, step (5) comprises: drying the carboxyl magnetic beads at room temperature for 1min-10min, and then adding eluent for elution.
in some embodiments, the method for recovering fragmented degraded DNA is characterized in that step (9) comprises:
uniformly mixing the solution containing the small fragment DNA finally obtained in the step (8) with a phosphate buffer solution, and adding the mixture into a centrifugal column;
adding a PE buffer solution into the centrifugal column, and cleaning the centrifugal column;
placing the empty centrifugal column into a centrifuge, centrifuging for 2min at full speed, and air drying at room temperature;
Adding eluent into the centrifugal column to elute the small fragment DNA.
the eluent is Tris buffer salt solution with the concentration of 3mM-15mM, and the pH value is 7.3-8.5.
in some embodiments, the agent I is formulated by: 3mM-15mM Tris buffer is dissolved in the excess water, and then the pH value of the solution is adjusted to 7.3-8.5 by HCl solution.
in some embodiments, after washing treatment in the carboxyl magnetic beads combined with the large fragments of degraded DNA, drying is performed at room temperature for 1min to 10min, and then 30uL to 80uL of reagent I is added.
In some embodiments, the elution temperature employed in step (9) is from 20 ℃ to 70 ℃.
in some embodiments, the amount of proteinase K used in step (9) is 100 uL.
In some embodiments, the buffer comprises buffer PB in an amount of 0.5mL to 3 mL.
in some embodiments, the carboxyl magnetic beads bound to large fragments of degraded DNA are placed in a Covaris fragmentation instrument for fragmentation to small fragments of degraded DNA.
in some embodiments, after all of the small fragment DNA is separated from the spin column, a volume of reagent I ranging from 15uL to 80uL is added to the spin column, and the spin column is eluted at 20 ℃ to 70 ℃.
In some embodiments, the method of recovering fragmented degraded DNA specifically comprises the steps of:
(1) 1ug of degraded DNA was taken and supplemented with water to the appropriate volume.
(2) Adding a certain volume of carboxyl magnetic beads into the DNA, uniformly mixing, and standing at room temperature for a proper time.
(3) the mixture was placed on a magnetic stand until clear, and the supernatant was transferred to a new 1.5mL centrifuge tube.
(4) The freshly prepared 80% ethanol was added to the carboxyl magnetic beads and washed 2 times.
(5) after drying at room temperature for a while, an appropriate volume of solution I is added for elution and left at room temperature for an appropriate time.
(6) The elution mixture was placed on a magnetic rack until clear, and the supernatant was transferred to a new 1.5mL centrifuge tube.
(7) the centrifuge tube from the previous step was transferred to a Covaris fragmentation instrument for fragmentation.
(8) Transferring the broken DNA into the centrifuge tube in the step (3), and fully and uniformly mixing.
(9) The appropriate volume of buffer PB was added to the mixed DNA solution, mixed well and added to the MinElute Column.
(10) Buffer PE was added to MinElute Column and the Column was washed.
(11) the column was emptied and dried at room temperature.
(12) Adding the solution I into a centrifugal column, and eluting DNA.
Adding water in the step (1) to a proper volume of 30uL-150 uL.
The using amount of the carboxyl magnetic beads in the step (2) is 20uL-100uL, and the incubation time is 5min-20 min.
and (3) drying at room temperature in the step (5) for 1-10 min.
in the step (5), the pH value of the reagent I is 7.3-8.5, the solvent is ultrapure water, and the solutes are the following substances with final concentration: tris 3mM-15mM, and HCl for adjusting pH value.
The adding volume of the reagent I in the step (5) is 30uL-80 uL.
The volume of the buffer PB added in the step (9) is 0.5mL-3 mL.
the adding volume of the reagent I in the step (12) is 15uL-80uL, and the elution temperature is 20-70 ℃. Preferably, in the step (12), the dosage of the proteinase K is 100 uL.
Preferably, in the step (1), water is added to the volume of 100 uL.
Preferably, the amount of the carboxyl magnetic beads in the step (2) is 60uL, and the incubation time is 10 min.
preferably, the room-temperature drying time in the step (5) is 3 min.
Preferably, in the step (5), the pH of the reagent I is 8.0, the solvent is ultrapure water, and the solutes are substances with the following final concentrations: tris 10mM, pH adjusted by addition of HCl.
Preferably, the volume of the reagent I added in the step (5) is 50 uL.
Preferably, the volume of the buffer PB added in the step (9) is 1.1 mL.
preferably, the volume of reagent I added in the step (12) is 30uL, and the elution temperature is 55 ℃.
The method for recovering the fragmented degraded DNA greatly improves the purification recovery rate of the fragmented degraded DNA and provides effective guarantee for the samples used for downstream molecular biology research.
the method for efficiently recovering fragmented degraded DNA only needs to use the existing carboxyl magnetic bead reagent and the PCR purification reagent in the market, has simple material source and low operation difficulty, and does not need special technical training and special instrument and equipment.
The technical scheme of the invention is further explained by combining the attached drawings and a plurality of embodiments and comparative examples.
The present invention will be further described with reference to specific examples, which are intended for illustrative purposes only and are not intended to be limiting.
before describing the examples, it is necessary to provide some remarks:
The reagent of different manufacturers and different batches can cause the difference of experimental results, and belongs to the normal phenomenon.
In a small-scale laboratory, in order to ensure the repeatability among parallel experiments, the reagent is recommended to be prepared, fully mixed and subpackaged so as to ensure the uniformity of the reagent in each experiment.
example 1
Preparation of reagents:
carboxylic magnetic Beads Ampure XP Beads (Beckman), Column QiaAmp MinElute Column (Qiagen), buffer PB (Qiagen), buffer PE (Qiagen), absolute ethanol (national medicine), Qubit dsDNA HS Assay (Life Technologies).
Reagent I: pH 8.0, solvent ultrapure water, solute containing the following final concentration substances, Tris buffer 10mM (national drug), HCl solution for pH adjustment.
The experimental operation process comprises the following steps:
(1) 1ug of degraded DNA was taken and water was added to 100 uL;
(2) Adding 60uL AmpureXP Beads into the degraded DNA in the step (1), uniformly mixing, and standing at room temperature for 10 min;
(3) Placing the mixed solution on a magnetic frame until the mixed solution is clear, and transferring supernatant liquid into a 1.5mL centrifuge tube;
(4) adding a freshly prepared 80 wt% ethanol solution into the lower layer mixed solution in the step (3), and cleaning for 2 times;
(5) Adding 50uL of reagent I for elution at room temperature for 3min, and standing at room temperature for 3 min;
(6) The elution mixture was placed on a magnetic rack until clear, and the supernatant was transferred to a new 1.5mL centrifuge tube.
(7) Transferring the centrifuge tube in the step (6) to a Covaris fragmentation instrument for fragmentation, wherein the fragmentation conditions are as follows:
| Intensity | 4 |
| Duty Cycle | 10％ |
| Cycle per Burst | 200 |
| Treatment Time(s) | 30 |
(8) And (4) transferring the broken DNA in the step (7) to the centrifuge tube in the step (3), and fully mixing the DNA and the centrifuge tube.
(9) Adding 1.1mL of buffer PB into the DNA solution mixed in the step (8), fully mixing, adding into MinElute Column, placing into a centrifuge, centrifuging at 8000rpm for 30s, and repeating the steps until all the DNA solution passes through the MinElute Column.
(10) To the MinElute Column was added 700uL of buffer PE, and the Column was washed 2 times.
(11) The column was emptied and air dried at room temperature for 3 min.
(12) add reagent I30 uL to MinElute Column center, incubate at 55 ℃ for 5min, centrifuge to get final product.
finally, the final product concentration was determined to be: 27.2 ng/ul.
example 2
preparation of reagents:
Carboxylic magnetic Beads Ampure XP Beads (Beckman), Column QiaAmp MinElute Column (Qiagen), buffer PB (Qiagen), buffer PE (Qiagen), absolute ethanol (national medicine), Qubit dsDNA HS Assay (Life Technologies).
Reagent I: pH 8.0, solvent ultrapure water, solute containing the following final concentration substances, Tris buffer 10mM (national drug), HCl solution for pH adjustment.
the experimental operation process comprises the following steps:
(1) 1ug of degraded DNA was taken and water was added to 100 uL;
(2) Adding 60uL AmpureXP Beads into the degraded DNA in the step (1), uniformly mixing, and standing at room temperature for 10 min;
(3) placing the mixed solution on a magnetic frame until the mixed solution is clear, and transferring supernatant liquid into a 1.5mL centrifuge tube;
(4) adding a freshly prepared 80 wt% ethanol solution into the lower layer mixed solution in the step (3), and cleaning for 2 times;
(5) adding 50uL of reagent I for elution at room temperature for 3min, and standing at room temperature for 3 min;
(6) the elution mixture was placed on a magnetic rack until clear, and the supernatant was transferred to a new 1.5mL centrifuge tube.
(7) Transferring the centrifuge tube in the step (6) to a Covaris fragmentation instrument for fragmentation, wherein the fragmentation conditions are as follows:
| Intensity | 4 |
| Duty Cycle | 10％ |
| Cycle per Burst | 200 |
| Treatment Time(s) | 30 |
(8) And (4) transferring the broken DNA in the step (7) to the centrifuge tube in the step (3), and fully mixing the DNA and the centrifuge tube.
(9) Adding 1.1mL of buffer PB into the DNA solution mixed in the step (8), fully mixing, adding into MinElute Column, placing into a centrifuge, centrifuging at 8000rpm for 30s, and repeating the steps until all the DNA solution passes through the MinElute Column.
(10) to the MinElute Column was added 700uL of buffer PE, and the Column was washed 2 times.
(11) The column was emptied and air dried at room temperature for 3 min.
(12) Add reagent I30 uL to MinElute Column center, incubate at 55 ℃ for 5min, centrifuge to get final product.
finally, the final product concentration was determined to be: 28.8 ng/ul.
Example 3
preparation of reagents:
Carboxylic magnetic Beads Ampure XP Beads (Beckman), Column QiaAmp MinElute Column (Qiagen), buffer PB (Qiagen), buffer PE (Qiagen), absolute ethanol (national medicine), Qubit dsDNA HS Assay (Life Technologies).
reagent I: pH 8.0, solvent ultrapure water, solute containing the following final concentration substances, Tris buffer 10mM (national drug), HCl solution for pH adjustment.
the experimental operation process comprises the following steps:
(1) 1ug of degraded DNA was taken and water was added to 100 uL;
(2) Adding 60uL AmpureXP Beads into the degraded DNA in the step (1), uniformly mixing, and standing at room temperature for 10 min;
(3) Placing the mixed solution on a magnetic frame until the mixed solution is clear, and transferring supernatant liquid into a 1.5mL centrifuge tube;
(4) Adding a freshly prepared 80 wt% ethanol solution into the lower layer mixed solution in the step (3), and cleaning for 2 times;
(5) adding 50uL of reagent I for elution at room temperature for 3min, and standing at room temperature for 3 min;
(6) The elution mixture was placed on a magnetic rack until clear, and the supernatant was transferred to a new 1.5mL centrifuge tube.
(7) Transferring the centrifuge tube in the step (6) to a Covaris fragmentation instrument for fragmentation, wherein the fragmentation conditions are as follows:
| Intensity | 4 |
| Duty Cycle | 10％ |
| Cycle per Burst | 200 |
| Treatment Time(s) | 30 |
(8) And (4) transferring the broken DNA in the step (7) to the centrifuge tube in the step (3), and fully mixing the DNA and the centrifuge tube.
(9) adding 1.1mL of buffer PB into the DNA solution mixed in the step (8), fully mixing, adding into MinElute Column, placing into a centrifuge, centrifuging at 8000rpm for 30s, and repeating the steps until all the DNA solution passes through the MinElute Column.
(10) to the MinElute Column was added 700uL of buffer PE, and the Column was washed 2 times.
(11) the column was emptied and air dried at room temperature for 3 min.
(12) Add reagent I30 uL to MinElute Column center, incubate at 55 ℃ for 5min, centrifuge to get final product.
Finally, the final product concentration was determined to be: 26.3 ng/ul.
Example 4
Preparation of reagents:
carboxylic magnetic Beads Ampure XP Beads (Beckman), Column QiaAmp MinElute Column (Qiagen), buffer PB (Qiagen), buffer PE (Qiagen), absolute ethanol (national medicine), Qubit dsDNA HS Assay (Life Technologies).
reagent I: pH 8.0, solvent ultrapure water, solute containing the following final concentration substances, Tris buffer 10mM (national drug), HCl solution for pH adjustment.
The experimental operation process comprises the following steps:
(1) 1ug of degraded DNA was taken and water was added to 100 uL;
(2) adding 60uL AmpureXP Beads into the degraded DNA in the step (1), uniformly mixing, and standing at room temperature for 10 min;
(3) Placing the mixed solution on a magnetic frame until the mixed solution is clear, and transferring supernatant liquid into a 1.5mL centrifuge tube;
(4) Adding a freshly prepared 80 wt% ethanol solution into the lower layer mixed solution in the step (3), and cleaning for 2 times;
(5) Adding 50uL of reagent I for elution at room temperature for 3min, and standing at room temperature for 3 min;
(6) The elution mixture was placed on a magnetic rack until clear, and the supernatant was transferred to a new 1.5mL centrifuge tube.
(7) transferring the centrifuge tube in the step (6) to a Covaris fragmentation instrument for fragmentation, wherein the fragmentation conditions are as follows:
| Intensity | 4 |
| Duty Cycle | 10％ |
| Cycle per Burst | 200 |
| Treatment Time(s) | 30 |
(8) And (4) transferring the broken DNA in the step (7) to the centrifuge tube in the step (3), and fully mixing the DNA and the centrifuge tube.
(9) Adding 1.1mL of buffer PB into the DNA solution mixed in the step (8), fully mixing, adding into MinElute Column, placing into a centrifuge, centrifuging at 8000rpm for 30s, and repeating the steps until all the DNA solution passes through the MinElute Column.
(10) to the MinElute Column was added 700uL of buffer PE, and the Column was washed 2 times.
(11) the column was emptied and air dried at room temperature for 3 min.
(12) Add reagent I30 uL to MinElute Column center, incubate at 55 ℃ for 5min, centrifuge to get final product.
Finally, the final product concentration was determined to be: 29.1 ng/ul.
Comparative example
Preparation of reagents:
Carboxylic magnetic beads QiaAmp MinElute Column (Qiagen), buffer PB (Qiagen), buffer PE (Qiagen), buffer EB (Qiagen), and Qubit dsDNA HS Assay (Life Technologies).
The experimental operation process comprises the following steps:
(1) 1ug of degraded DNA was taken to 50uL with water and transferred to a Covaris fragmentation instrument for fragmentation under the following conditions:
| Intensity | 4 |
| Duty Cycle | 10％ |
| Cycle per Burst | 200 |
| Treatment Time(s) | 30 |
(2) Adding 250uL buffer solution PB into the broken DNA solution, fully mixing, adding into MinElute Column, placing into a centrifuge, centrifuging at 8000rpm for 30s, and repeating the steps until all the DNA solution passes through the MinElute Column.
(3) To the MinElute Column was added 700uL of buffer PE, and the Column was washed 2 times.
(4) the column was emptied and air dried at room temperature for 3 min.
(5) Buffer EB 30uL was added to the MinElute Column center, incubated at 55 ℃ for 5min, and centrifuged to obtain the final product.
Finally, the final product concentration was determined to be: 5.2 ng/ul.
As can be seen from the drawings of FIG. 1 and FIG. 2, the method for efficiently recovering fragmented degraded DNA of the present invention has the advantages of simple reagent components, convenient operation, low cost, no need of special equipment, and capability of improving the fragmentation recovery rate of the degraded DNA, wherein the recovery rate is far higher than the existing fragmentation recovery rate of the DNA. Therefore, the invention can effectively improve the yield of degraded DNA and fragmentation, and greatly improve the problems encountered by scientific researchers when processing degraded DNA for downstream molecular biological research.
it should be understood that the above-mentioned embodiments are merely illustrative of the technical concepts and features of the present invention, which are intended to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and therefore, the protection scope of the present invention is not limited thereby. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (7)
1. a method for recovering fragmented degraded DNA, comprising:
(1) Dissolving degraded DNA in water to form a degraded DNA solution, wherein the concentration of the degraded DNA in the degraded DNA solution is 1 mug/30 mug-1 mug/150 mug;
(2) Adding carboxyl magnetic beads into a degraded DNA solution, uniformly mixing, and incubating at room temperature to ensure that the carboxyl magnetic beads are fully combined with large-fragment DNA and small-fragment DNA is retained in the solution, wherein the size of the large-fragment DNA is more than 2000bp, and the size of the small-fragment DNA is 200 bp-2000 bp;
(3) Settling the carboxyl magnetic beads by using the action of a magnetic field until a mixed solution is clarified, separating the carboxyl magnetic beads, and collecting a clarified solution containing small-fragment DNA;
(4) washing the carboxyl magnetic beads;
(5) Drying the carboxyl magnetic beads at room temperature, eluting with eluent, and standing at room temperature;
(6) Clarifying the elution mixed solution obtained in the step (5) by using the action of a magnetic field, and separating out the clarified solution;
(7) transferring the clear solution separated in the step (6) into a fragmenting instrument for fragmentation treatment, so that large fragment DNA is broken into small fragment DNA;
(8) Combining the solution containing small fragment DNA obtained in the step (7) with the clarified solution containing small fragment DNA obtained in the step (3);
(9) Uniformly mixing the solution containing the small fragment DNA finally obtained in the step (8) with a phosphate buffer solution, and adding the mixture into a centrifugal column;
Adding a PE buffer solution into the centrifugal column, and cleaning the centrifugal column;
Placing the empty centrifugal column into a centrifuge, centrifuging for 2min at full speed, and air drying at room temperature;
Adding eluent into the centrifugal column to elute the small fragment DNA.
2. The method for recovering fragmented degraded DNA according to claim 1, characterized in that step (2) comprises: adding the carboxyl magnetic beads into the solution for degrading DNA, uniformly mixing, and incubating at room temperature for 5-20 min to ensure that the carboxyl magnetic beads are fully combined with the large-fragment DNA.
3. The method for recovering fragmented degraded DNA according to claim 1, characterized in that step (4) comprises: and washing the carboxyl magnetic beads by using an ethanol solution with the concentration of 80 v/v%.
4. The method for recovering fragmented degraded DNA according to claim 1, characterized in that step (5) comprises: drying the carboxyl magnetic beads at room temperature for 1min-10min, and then adding eluent for elution.
5. The method for recovering fragmented degraded DNA according to claim 1, characterized in that: the eluent is Tris buffer salt solution with the concentration of 3mM-15mM, and the pH value is 7.3-8.5.
6. The method for recovering fragmented degraded DNA according to claim 1, characterized in that: the elution temperature used in step (9) is 20-70 ℃.
7. the method for recovering fragmented degraded DNA according to claim 1, characterized in that: the amount of the eluent used in the step (9) was 100. mu.L.
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