CN114790454A - Kit and method for high-throughput full-automatic chicken blood DNA efficient extraction - Google Patents
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Abstract
The invention provides a kit and a method for high-throughput full-automatic chicken blood DNA efficient extraction. The kit comprises at least one of a blood cell lysate and an enhanced plate lysate, a binding solution, a washing solution I, a washing solution II and an eluent; the kit is based on a magnetic bead method, full-automatic large-batch extraction of complete genome DNA from chicken blood is realized in a one-step cracking mode, the purity and concentration of the obtained DNA are good, and the kit can be used for high-throughput experiments. The invention adopts a full-automatic workstation Beckman NX P The throughput can be up to 192 samples at a time. The genomic DNA extracted by the method has better purity, yield and strip integrity, the cost is greatly reduced on the premise of ensuring high flux and high efficiency, and the method has popularization value in animal production research and application.
Description
Technical Field
The invention relates to the field of molecular biology, and relates to a kit and a method for high-throughput full-automatic chicken blood DNA efficient extraction.
Background
The molecular biology technology is used for researching complex genomes, particularly complex molecular experiments such as DNA library construction, sequencing, Southern blotting, AFLP amplification fragment length polymorphism, fluorescence quantitative PCR and the like, and the requirement on preparing pure high molecular weight DNA is severe. For example, the quality of DNA including concentration, purity, integrity, and accurate quantification of DNA in sequencing technologies can directly affect the data throughput and accuracy of sequencing.
The feather stem cuticle of chicken is thick, and is not easy to digest, and simultaneously protein content is higher, and DNA content is less, is not suitable for being used as the raw material of DNA extraction, and chicken blood is generally used as the raw material of extraction in animal production, but chicken blood is relatively harsh to the preservation mode, and needs to be preserved at low temperature by anticoagulant, otherwise the condition of blood coagulation or hemolysis is easily caused, blood coagulation is easily formed in the large-scale sampling process in production, the DNA extraction effect of blood coagulation is poor, and the expression is that DNA concentration, purity and integrity are poor, and simultaneously, the accuracy of DNA quantification is also greatly influenced. The traditional phenol-chloroform extraction method and the centrifugal column method have good DNA extraction effect, but the efficiency of mass extraction is low, and automation cannot be realized, while the magnetic bead method is a genome DNA extraction method which is developed more quickly in recent years, and automation can be realized on the premise of ensuring the extraction effect, but the traditional phenol-chloroform extraction method and the centrifugal column method mainly adopt 48 flux and 96 flux in the market at present, and still have larger space for improving efficiency. Meanwhile, the price of the nucleic acid extraction kit on the market is high, the nucleic acid extraction cost of a single sample is higher than 10 yuan, and great limitation is formed on large-scale popularization and application in animal production.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a kit and a method for high-throughput full-automatic chicken blood DNA efficient extraction. The kit is based on a magnetic bead method, full-automatic large-scale extraction of complete genome DNA is performed from chicken blood in a one-step cracking mode, the obtained DNA has good purity and concentration, and the kit can be used for high-throughput molecular biology experiments.
In order to realize the purpose, the technical scheme is as follows: a kit for high-efficiency extraction of high-throughput full-automatic chicken blood DNA comprises at least one of a hemocyte lysate and an enhanced plate lysate, a binding solution, a washing solution I, a washing solution II and an eluent;
the blood cell lysate comprises Tris, EDTA, guanidine hydrochloride, NaCl and Triton X-100, and the enhanced blood cell lysate comprises Tris, EDTA, guanidine hydrochloride, guanidine isothiocyanate, NaCl, Triton X-100 and 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid;
the binding solution comprises PEG600 and NaCl;
the washing solution I comprises guanidine hydrochloride, NaCl, Tris, EDTA and isopropanol, and the washing solution II comprises an ethanol water solution;
the eluent comprises Tris-HCl and EDTA.
Preferably, the blood cell lysate comprises 4-8M guanidine hydrochloride, 150-200 mM NaCl, 10-50 mM EDTA, 50-100 mM Tris, 1-4% Triton X-100, and the pH is 7.0-9.0; more preferably, the blood cell lysate comprises 6M guanidinium hydrochloride, 150mM NaCl, 10mM EDTA, 50mM Tris, 1% Triton X-100, pH 7.8;
preferably, the enhanced blood cell lysate comprises 4-8M guanidine hydrochloride, 3-6M guanidine isothiocyanate, 150-200 mM NaCl, 10-50 mM EDTA, 50-100 mM Tris, 2-4% Triton X-100, 30-100 g/L3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid, and the pH is 7.0-9.0; more preferably, the enhanced blood cell lysate comprises 6M guanidinium hydrochloride, 4M guanidinium isothiocyanate, 200mM NaCl, 10mM EDTA, 50mM Tris, 4% Triton X-100, 50 g/L3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid, and pH 7.8. The enhanced lysis solution is required for the blood coagulation nucleic acid extraction.
Preferably, the binding solution comprises 5-25% by mass of PEG6000 and 0.25-2.5M NaCl, and the PH value is 7.0-9.0; more preferably, the binding solution comprises 10% by mass of PEG6000 and 1.25M NaCl, and the pH value is 7.8.
Preferably, the washing solution I comprises 2-6M guanidine hydrochloride, 2-5 mM EDTA, 10-50 mM Tris, 0.15% by mass of HCl, 20-50% by volume of isopropanol and 7.0-9.0 of PH; preferably, the washing solution I comprises 4M guanidine hydrochloride, 2.5mM EDTA, 20mM Tris, 0.15% by mass of HCl, 25% by volume of isopropanol and pH 7.8;
the washing liquid II comprises an ethanol solution with the volume fraction of 70-75%; preferably, the washing solution II comprises ethanol solution with the volume fraction of 70%.
Preferably, the eluent comprises 10-100 mM Tris-HCl and 0.1-1 mM EDTA, and the pH value is 7.0-9.0; preferably, the eluent comprises 10mM Tris-HCl, 1mM EDTA, and pH 8.0.
Preferably, the kit further comprises magnetic beads, water and absolute ethyl alcohol; more preferably, the concentration of the magnetic beads is 50-200 mg/mL, and the magnetic beads are hydroxyl magnetic beads or silicon-based magnetic beads. The magnetic beads can use hydroxyl magnetic beads or silicon-based magnetic beads used for nucleic acid purification on the market, the silicon-based magnetic beads used for Meiji, the hydroxyl magnetic beads used for Changchun Chong and the Ampure magnetic beads used for Beckman are matched with a self-developed nucleic acid extraction kit, and the DNA effect is good.
The invention provides application of the kit in full-automatic chicken blood DNA high-efficiency extraction.
In order to achieve high-flux high-efficiency extraction of high-quality chicken blood DNA, a full-automatic nucleic acid extraction scheme is adopted. Preferably, the full automation is achieved by using a fully automated workstation Beckman NX P The realization is that the board position of the workstation is slightly adjusted, and the method is also suitable for other types of workstations.
The invention provides a method for efficiently extracting high-throughput full-automatic chicken blood DNA, which adopts the kit to extract the chicken blood DNA and comprises the following steps:
(1) sucking/picking chicken blood: for anticoagulation (blood flows smoothly without obvious coagulum/silk) with good anticoagulation effect, the layered anticoagulation is inverted up and down and mixed evenly, then a pipettor is used for sucking the middle layer blood, and the middle layer blood is diluted by ultrapure water; for the blood clot/blood clot silk, the blood clot/blood clot silk at the blood coagulation fine head part of the chicken is picked by a needle head of an injector and diluted by ultrapure water;
(2) digestion of chicken blood: adding the blood cell lysate and the proteinase K into the solution obtained in the step 1, fully and uniformly mixing, and digesting for 0.5h-18h at 56 ℃;
(3) binding of DNA: adding a binding solution, absolute ethyl alcohol and magnetic beads into the solution obtained in the step (2) for oscillation binding, moving the solution to a magnetic rack for DNA adsorption, and absorbing and discarding the waste liquid;
(4) washing of the DNA: firstly, adding a washing solution I to wash the magnetic beads twice, and absorbing and discarding waste liquid; adding a washing solution II for washing twice, sucking and discarding the waste liquid, and drying at 56 ℃;
(5) elution of DNA: adding TE for resuspending magnetic beads, oscillating at 56 ℃ for 10-15 min at high speed, transferring to a magnetic frame for magnetic bead adsorption, and absorbing the DNA-containing supernatant to obtain a DNA-containing solution.
Preferably, the step (1) of sucking/picking chicken blood comprises the following steps: for anticoagulation with a good anticoagulation effect, after the layered anticoagulation is inverted and mixed up and down, a pipettor is used for sucking 2.5-5 mu L of middle-layer blood, and the middle-layer blood is diluted to 100 mu L by ultrapure water; for the blood clot/blood clot silk, the blood clot/blood clot silk of the chicken blood clot fine head part is picked up by a needle head of a 1mL syringe and diluted to 100 mu L by ultrapure water; the step 1) uses a full-automatic workstation Beckman NX P Carrying out ultrapure water adding operation;
adding 200 mu L of blood cell lysate and 20 mu L of 20mg/mL proteinase K in the step (2), and digesting for more than 0.5h at 56 ℃; the step (2) uses a full-automatic workstation Beckman NX P Adding, uniformly mixing and incubating hemocyte lysate and protease PK;
adding 100 mu L of binding solution, 300 mu L of absolute ethyl alcohol and 10 mu L of magnetic beads into the step (3), intermittently oscillating for 8-10min, transferring the mixture to a magnetic frame to adsorb DNA for 1min, and absorbing and discarding waste liquid, wherein the step (3) uses a full-automatic workstation Beckman NX P And (5) carrying out operation.
Adding 600 mu L of washing liquid I in the step (4), oscillating for 1min, transferring to a magnetic frame for 1min, absorbing and discarding the waste liquid, and repeating the step 1 time; adding washing solution II, oscillating for 1min, transferring to a magnetic frame for 1min, absorbing and discarding the waste liquid, and repeating the above steps for 1 time; airing; the step (4) uses a full-automatic workstation Beckman NX P Carrying out operation;
adding 120 mu L of eluent TE into the step (5), oscillating for 15min, transferring to a magnetic frame for 2-5min, absorbing the DNA-containing supernatant to a DNA sample storage plate, and storing at-20 ℃; the step (5) uses a full-automatic workstation Beckman NX P And (5) carrying out operation.
Preferably, the method further comprises quality inspection of the DNA: the DNA concentration is automatically or manually detected by a microplate reader or Nanodrop8000, and the premixed DNA and the Loading buffer are used for electrophoresis detection.
In the step (1), when blood coagulation is picked, the part needing to be picked needs attention to be the thin-head part of the blood clot, and the part below the blood clot can be lightly picked by a needle head to be turned over. Preferably, the blood clot/blood filament picked is a ground peanut sized blood clot/blood filament.
In the step (2), the specific process of chicken blood digestion is as follows: cracking and digesting by using a 96-hole deep-hole plate, adding 200 mu L of blood cell lysate and 20 mu L of proteinase K (20mg/mL) into each hole, fully oscillating, uniformly mixing and centrifuging with a chicken blood sample, and cracking at 56 ℃ for 0.5-18 h, wherein the period can be oscillating and uniformly mixing for 2-4 times.
In the step (3), the specific process of DNA combination is as follows: 2 sample digestion plates to be extracted, 2 96-well magnetic bead plates (100. mu.L of DNA binding solution and 10. mu.L of magnetic beads), 2 column-type magnetic racks, and 2 cassettes of tips; using Beckman NX p The oscillation module oscillates the magnetic bead plate in sequence, the liquid transfer arm transfers 196 sample digestive juice to corresponding hole sites in the corresponding 96-hole magnetic bead plate respectively, the high-speed oscillation is carried out for 8min, the DNA is fully contacted and combined with the magnetic beads, and Beckman NX p The magnetic bead plate is transferred to the magnetic frame by the gripper and stands still for 1min, and the liquid-transferring arm sucks waste liquid to the sample digestion plate.
In the step (4), the specific process of DNA washing is as follows: step 5, magnetic bead plate, 2 column type magnetic racks, 4 box suction heads, 4 waste liquid plates and 2 liquid tanks (respectively filled with washing liquid I)And 70% ethanol); using Beckman NX p Transferring 600 mu L of washing liquid I to the magnetic bead plate obtained in the step 5 by using the liquid transferring arm, oscillating and washing for 1min, transferring the magnetic bead plate to a magnetic rack by using a gripper, standing for 20s, transferring waste liquid to a waste liquid plate after the magnetic beads are fully adsorbed, and repeatedly washing Wash A once; using Beckman NX p Transferring 600 mu L of 70% EtOH to the magnetic bead plate obtained in the step 5 by using the liquid transfer arm, oscillating and washing for 1min, transferring the magnetic bead plate to a magnetic rack by using a gripper, standing for 20s, transferring waste liquid to a waste liquid plate after the magnetic beads are fully adsorbed, and repeatedly washing 70% EtOH for one time; transferring the magnetic bead plate to an incubation module by the gripper, and drying for 2-5min at 56 ℃;
in the step (5), the specific process of DNA elution is as follows: step 6, magnetic bead plates, 2 column type magnetic racks, 2 suction heads, 2 liquid tanks (filled with TE and Gel loading buffers), 2 DNA storage plates, 2 96-hole enzyme label plates and 2 96-hole sample spotting plates (used for samples of subsequent electrophoresis); using Beckman NX p And (3) transferring 120 mu LTE to the magnetic bead plate obtained in the step (6) by using the liquid transfer arm, carrying out high-speed oscillation elution for 15min, transferring the magnetic bead plate to a magnetic rack by using a hand grip, standing for 2-5min, and slowly absorbing 95 mu L of DNA-containing supernatant to a DNA storage plate after the magnetic beads are fully adsorbed.
In the step (6), the specific process of DNA quality inspection is as follows: transferring 25 mu LDNA to a 96-hole enzyme label plate (pre-packaged with 25 mu L of water), and uniformly mixing 2 mu LDNA to a 96-hole spotting plate (pre-packaged with 8 mu L of loading buffer), wherein the enzyme label plate can be directly placed into an enzyme label instrument to detect the DNA concentration or can be rapidly and manually detected by using Nanodrop8000, and the spotting plate can be rapidly subjected to sample loading electrophoresis by using an eight-channel arraying gun.
Compared with the prior art, the invention has the following advantages and effects:
the reagent has extremely low cost, and the preparation method is simple and controllable, and can be popularized and applied on a large scale in animal production research.
The invention solves the problems of difficult blood coagulation extraction, poor purity and inaccurate concentration detection of the chicken, and improves the quality of the chicken blood.
The invention solves the problems that the DNA extraction concentration has large difference in height, homogenization operation is needed, time and cost are consumed, the DNA concentration extracted by the invention has high homogenization degree, independent sample dilution is not needed, and the DNA can be directly diluted uniformly according to the experiment requirements for downstream experiments.
The invention solves the problems that the DNA used for high-throughput detection needs to be subjected to Qubit detection, time and labor are wasted, cost is wasted, and operation is required by professional personnel, and the ultraviolet spectrophotometry value of using the Nanodrop can be directly used for subsequent experiments by improving the quality of the DNA.
The invention adopts a full-automatic workstation Beckman NX P The highest throughput can reach 192 samples at a time. The genomic DNA extracted by the method has better purity, yield and strip integrity, the cost is greatly reduced on the premise of ensuring high flux and high efficiency, and the method has popularization value in animal production research and application.
Drawings
FIG. 1 is a schematic diagram of a layout of a fully automated workstation for high-throughput nucleic acid extraction according to the present invention.
FIG. 2 shows the result of agarose gel electrophoresis of genomic DNA extracted from chicken normal blood in example 2. Note: m22120 bpmarker, 1 and 2 of 1 mu L chicken blood DNA, 2 and 4 of 2 mu L chicken blood DNA, 5 and 6 of 3 mu L chicken blood DNA, 7 and 8 of 4 mu L chicken blood DNA, and 9 and 10 of 5 mu L chicken blood DNA.
FIG. 3 shows the result of agarose gel electrophoresis of genomic DNA extracted from chicken blood coagulation in example 4. Note: m22120 bpmarker, 1 and 2 are 1 muL of chicken blood coagulation DNA, 3 and 4 are 2 muL of chicken blood coagulation DNA, 5 and 6 are 3 muL of chicken blood coagulation DNA, and 7 and 8 are 5 muL of normal chicken blood DNA.
FIG. 4 is a schematic diagram of the nucleic acid extraction process of the present invention.
FIG. 5 is a diagram showing the library construction effect of the chicken blood genomic DNA extracted according to the present invention. The abscissa indicates the length of the band and the ordinate indicates the concentration of the band. Sample: and (3) sampling.
Detailed Description
The present invention will be further illustrated by the following examples, but the embodiments of the present invention are not limited thereto.
Example 1 verification of the efficacy and results of the nucleic acid extraction kit.
TABLE 1 formulation of nucleic acid extraction kit
The kit is used for extracting DNA from normal chicken blood, the formula of the kit is shown as an example 1 in a table 1, and the steps are as follows:
(1) in order to obtain the chicken blood in a normal state under the condition of mass sampling, two modes of increasing the dosage of the anticoagulant and reducing the volume of the collected chicken blood can be adopted. 120 mu L of EDTA-2Na anticoagulant (15mg/mL, PH 4.5-8.0) can effectively anticoagulate 1mL of chicken blood, a throat sprayer is used for spraying the EDTA-2Na anticoagulant into a centrifuge tube to obtain an anticoagulation tube, and after the chicken blood is injected into the anticoagulation tube, the key point is that the chicken blood and the anticoagulant are quickly contacted and uniformly mixed. Sucking 1-5 μ L of normal chicken blood (without coagulation/hemolysis state) into corresponding wells of a 96-well plate.
(2) Using Beckman NX P The workstation carries out chicken blood digestion and cracking steps: adding 200 μ L of lysis solution and 20 μ L of proteinase K (20mg/mL) into the sample well, shaking at 1000rpm for 1min, and incubating at 56 deg.C for 30min to completely denature the protein.
(3) Using Beckman NX P The workstation carries out chicken blood DNA combination: and (2) adding 100 mu L of binding solution and 300 mu L of absolute ethyl alcohol (the absolute ethyl alcohol: the lysate + the sample diluted in the step (1): 1), placing 10 mu L of magnetic beads into the sample hole, intermittently oscillating at 1000rpm for 8-10min, and interactively operating the two sample plates by using the operation standing time, so that the whole operation time can be saved, transferring the sample plates to a magnetic frame to adsorb DNA, and discarding the waste liquid.
(4) Using Beckman NX P The workstation carries out a chicken blood DNA washing step: adding 600 mu L of washing solution I into the sample hole, oscillating and uniformly mixing for 1min at 1000rpm, transferring to a magnetic frame to adsorb DNA for 1min, absorbing waste liquid, and repeating all the operations once; adding 600 mu L of washing solution II into the sample hole, shaking and uniformly mixing at 1000rpm for 1min, transferring to a magnetic frame to adsorb DNA for 1min, absorbing and discarding the waste liquid, and repeating all the operations once; can use the operation standing time to interactively operate the two sample plates, canThe overall operating time is saved. The sample plate was dried at 56 ℃ for 2-5 min.
(5) Using Beckman NX P The workstation carries out chicken blood DNA elution: adding 120 mu L of TE into the sample hole, oscillating at 1200rpm for 15min, transferring to a magnetic rack, standing for 2-5min, sucking 95 mu L of supernatant to a DNA storage plate, sucking 25 mu L of supernatant to an ELISA plate, and sucking 2 mu L of supernatant to a sample plate.
(6) DNA quality testing step: the concentration of DNA in the ELISA plate can be automatically detected directly by an ELISA reader, and the DNA in a DNA preservation plate can also be manually detected by using Nanoprop 8000, wherein the sample loading amount of each sample is 2 mu L. The electrophoresis was performed directly using the DNA in the spotting plate premixed with the loading buffer, and the results are shown in Table 2.
(7) Using Beckman NX p The table layout of the workstation for carrying out the high-throughput avian blood nucleic acid extraction method is shown in FIG. 1.
TABLE 2 example 1 extraction of genomic DNA concentration, Total amount and purity from Normal Chicken blood
The volume ratios of the absolute ethyl alcohol in the step (3) to the sample diluted in the lysis solution and the step (1) in the examples 1 to 4 and the comparative examples 2 to 4 are absolute ethyl alcohol: lysate + diluted sample ═ 1: 1; in comparative example 1, the volume ratio of the absolute ethyl alcohol to the lysis solution in the step (3) and the sample diluted in the step (1) is absolute ethyl alcohol: lysate + diluted sample 3: 7;
comparative examples 1 to 4 were each 3ul of the blood coagulation sample 1 (1) and 3ul of the normal blood sample 2 (2), the extraction procedure was the same as above, and the results of measuring concentration and purity are shown in the following table 3:
TABLE 3 concentration, total amount and purity of genomic DNA extracted from the kits of comparative examples 1 to 4
Example 2
The method is used for extracting chicken blood DNA based on an autonomously developed DNA magnetic bead method extraction kit, the formula of the kit is shown in an embodiment 2 in a table 1, and the steps are as follows:
(1) large scale production of blood samples inevitably produces individually coagulated samples. And sucking 2-5 mu L of partial flowing blood of the blood coagulation sample into a corresponding hole of the 96-pore plate.
(2) The remaining procedure was the same as in example 1.
The results are shown in table 4 and fig. 2.
TABLE 4 example 2 extraction of genomic DNA concentration, Total amount and purity from Chicken blood coagulation samples
Example 3
The method is improved and optimized to extract chicken blood coagulation DNA based on example 2, and according to the results in table 4, we find that the DNA extracted by using partial flowing blood of a blood coagulation sample is poor in quality and low in concentration, and cannot meet the experimental requirements, probably because the main component of the flowing blood is plasma, blood cells are few, and meanwhile, hemolysis can exist, so that DNA heredity is little or even none, and the sampling step is improved.
The specific steps are changed as follows:
(1) selecting a proper amount of blood clots at the coagulation fine head of the chicken by using the cross section of a needle head of a 1mL syringe;
(2) the remaining steps were the same as in example 2.
The results are shown in Table 5.
TABLE 5 example 3 extraction of genomic DNA concentration, Total amount and purity from Chicken blood coagulation samples
Example 4
The method is improved and optimized based on example 3 to extract the chicken blood coagulation DNA, and according to the results in table 5, we find that the concentration of the DNA extracted by using the blood clot of the blood coagulation sample is improved, but the concentration and the purity do not meet the experimental requirements, and the digestion and lysis steps are improved probably because the chicken blood sample is not completely lysed.
The specific steps are changed as follows:
(1) the sampling of blood coagulation in the step 1 is the same as that in the example 3, and the sampling of normal blood is the same as that in the example 1, wherein the samples 7-4 and 8-4 are normal blood and can be sampled normally, and the rest samples 1-6 are blood coagulation.
(2) And 2, adding 200 mu L of enhanced blood cell lysate and 20 mu L of PK protease, oscillating and mixing uniformly, putting the mixture into a container at 56 ℃, oscillating and incubating for more than 1h, oscillating for 2-4 times, and performing the same steps as in the example 3.
As can be seen from Table 1, the concentration and purity of the chicken blood genome DNA in example 1 are still good, which indicates that the quality of the normal chicken blood DNA sample extracted by the method is good and can meet the requirement of high-throughput detection. In the example 2, the same extraction method as that of normal blood is adopted for extracting the chicken blood coagulation, the sampling type is the flowing blood in the blood coagulation, and the flowing blood is considered to have the main component of plasma, so that the DNA content is low, the overall extraction effect is influenced, the DNA quality is poor, and the batch repeatability is poor. On the basis of the embodiment 2, the adjustment of the sampling type is made, the sampling type is changed into that a needle head is used for picking a proper amount of blood coagulation parts, the DNA extraction effect is improved, the concentration is greatly improved, but the purity is still poor, considering that the blood coagulation is equivalent to a tissue block, if the condition that the lysis is not complete can be caused only by using a mild lysis solution, a reinforced plate blood cell lysis solution is needed to be used for completely and gradually denaturing the protein, the test result is shown in table 6 and figure 3, the DNA concentration, the purity and the total quantity are good, the DNA integrity is good, the concentration is also increased in a gradient manner along with the increase of the chicken blood volume, the sampling volume can be adjusted according to the experiment requirement to obtain the required concentration, the concentration equalization can be favorably realized, and the DNA or the blood coagulation of normal chicken blood can be extracted by using the method of the embodiment 4 (the operation flow is shown in figure 4), the DNA with higher quality and better repeatability and stability, the requirement of high-flux detection is met, and the effect is shown in figure 5.
TABLE 6 example 4 extraction of genomic DNA concentration, Total amount and purity from Chicken blood clotting/Normal blood samples
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A kit for high-efficiency extraction of high-throughput full-automatic chicken blood DNA is characterized by comprising at least one of a hemocyte lysate and an enhanced plate lysate, a binding solution, a washing solution I, a washing solution II and an eluent;
the blood cell lysate comprises Tris, EDTA, guanidine hydrochloride, NaCl and Triton X-100, and the enhanced blood cell lysate comprises Tris, EDTA, guanidine hydrochloride, guanidine isothiocyanate, NaCl, Triton X-100 and 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid;
the binding solution comprises PEG600 and NaCl;
the washing solution I is prepared from guanidine hydrochloride, NaCl, Tris, EDTA and isopropanol, and the washing solution II comprises an ethanol water solution;
the eluent comprises Tris-HCl and EDTA.
2. The kit of claim 1, wherein the blood cell lysate comprises 4-8M guanidine hydrochloride, 150-200 mM NaCl, 10-50 mM EDTA, 50-100 mM Tris, 1-4% Triton X-100, PH 7.0-9.0; preferably, the blood cell lysate comprises 6M guanidinium hydrochloride, 150mM NaCl, 10mM EDTA, 50mM Tris, 1% Triton X-100, pH 7.8;
the enhanced blood cell lysate comprises 4-8M guanidine hydrochloride, 3-6M guanidine isothiocyanate, 150-200 mM NaCl, 10-50 mM EDTA, 50-100 mM Tris, 2% -4% Triton X-100, and 30-100 g/L3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid, wherein the pH value is 7.0-9.0; preferably, the enhanced blood cell lysate comprises 6M guanidine hydrochloride, 4M guanidine isothiocyanate, 200mM NaCl, 10mM EDTA, 50mM Tris, 4% Triton X-100, 50 g/L3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid, pH 7.8.
3. The kit according to claim 1, wherein the binding solution comprises 5-25% by mass of PEG6000, 0.25-2.5M NaCl, and pH 7.0-9.0; preferably, the binding solution comprises 10% by mass of PEG6000 and 1.25M NaCl, and the pH value is 7.8.
4. The kit according to claim 1, wherein the washing solution I comprises 2-6M guanidine hydrochloride, 2-5 mM EDTA, 10-50 mM Tris, 0.15% by mass HCl, 20-50% by volume isopropanol, and pH 7.0-9.0; preferably, the washing solution I comprises 4M guanidine hydrochloride, 2.5mM EDTA, 20mM Tris, 0.15% by mass of HCl, 25% by volume of isopropanol and pH 7.8;
the washing liquid II comprises an ethanol solution with the volume fraction of 70-75%; preferably, the washing solution II comprises ethanol solution with the volume fraction of 70%.
5. The kit according to claim 1, wherein the eluent comprises 10-100 mM Tris-HCl, 0.1-1 mM EDTA, and pH 7.0-9.0; preferably, the eluent comprises 10mM Tris-HCl, 1mM EDTA, and pH 8.0.
6. The kit of claim 1, wherein the kit further comprises magnetic beads, water, and absolute ethanol; preferably, the concentration of the magnetic beads is 50-200 mg/mL, and the magnetic beads are hydroxyl magnetic beads or silicon-based magnetic beads.
7. The application of the kit of any one of claims 1-6 in full-automatic chicken blood DNA high-efficiency extraction.
8. A method for high-efficiency extraction of high-throughput full-automatic chicken blood DNA, which is characterized in that the method adopts the kit as claimed in any one of claims 1 to 6 to extract the chicken blood DNA, and comprises the following steps:
(1) sucking/picking chicken blood: for anticoagulation with better anticoagulation effect, the layered anticoagulation is inverted up and down and mixed evenly, the middle layer blood is sucked by a pipettor, and diluted by ultrapure water; for the blood clot/blood clot silk, the needle head of the syringe is used for picking the blood clot/blood clot silk at the blood clot thin head part of the chicken, and the blood clot/blood clot silk is diluted by ultrapure water;
(2) digestion of chicken blood: adding the blood cell lysate and the proteinase K into the solution obtained in the step 1, fully and uniformly mixing, and digesting for 0.5-18 h at 56 ℃;
(3) binding of DNA: adding a binding solution, absolute ethyl alcohol and magnetic beads into the solution obtained in the step (2) for oscillation binding, moving the solution to a magnetic rack for DNA adsorption, and absorbing and discarding the waste liquid;
(4) washing of DNA: firstly, adding a washing solution I to wash the magnetic beads twice, and absorbing and discarding waste liquid; adding a washing solution II for washing twice, sucking and discarding the waste liquid, and drying at 56 ℃;
(5) elution of DNA: and adding TE for resuspending the magnetic beads, oscillating at 56 ℃ for 10-15 min at high speed, transferring to a magnetic frame for adsorbing the magnetic beads, and absorbing the supernatant to obtain a solution containing DNA.
9. The method of claim 8, wherein the step (1) of sucking/picking the chicken blood comprises: for anticoagulation with a good anticoagulation effect, the layered anticoagulation is inverted up and down and mixed uniformly, then a pipette is used for sucking 2.5-5 mu L of middle-layer blood, and the middle-layer blood is diluted to 100 mu L with ultrapure water; for coagulum/coagulopathy, the chicken thrombocyte head is picked up by using a needle of a 1mL syringeBlood clot/blood streak, diluted to 100 μ L with ultrapure water; the step 1) uses a full-automatic workstation Beckman NX P Carrying out the operation of adding ultrapure water;
adding 200 mu L of blood cell lysate and 20 mu L of 20mg/mL proteinase K in the step (2), and digesting for more than 0.5h at 56 ℃; the step (2) uses a full-automatic workstation Beckman NX P Adding, uniformly mixing and incubating hemocyte lysate and protease PK;
adding 100 mu L of binding solution, 300 mu L of absolute ethyl alcohol and 10 mu L of magnetic beads into the step (3), intermittently oscillating for 8-10min, transferring the mixture to a magnetic rack for adsorbing DNA for 1min, and absorbing and discarding waste liquid, wherein a full-automatic workstation Beckman NX is used in the step (3) P Carrying out operation;
adding 600 mu L of washing liquid I in the step (4), oscillating for 1min, transferring to a magnetic frame for 1min, sucking and discarding waste liquid, and repeating the step 1 time; adding washing solution II, oscillating for 1min, transferring to a magnetic frame for 1min, absorbing and discarding the waste liquid, and repeating the above steps for 1 time; airing; the step (4) uses a full-automatic workstation Beckman NX P Carrying out operation;
adding 120 mu L of eluent TE into the step (5), oscillating for 15min, transferring to a magnetic frame for 2-5min, absorbing the DNA-containing supernatant to a DNA sample storage plate, and storing at-20 ℃; the step (5) uses a full-automatic workstation Beckman NX P And (5) carrying out operation.
10. The method of claim 8, further comprising quality testing of the DNA: the DNA concentration is automatically or manually detected by a microplate reader or Nanodrop8000, and the premixed DNA and the Loading buffer are used for electrophoresis detection.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117230060A (en) * | 2023-11-13 | 2023-12-15 | 苏州为度生物技术有限公司 | Pre-lysis solution, kit and method for extracting genome DNA |
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| WO1996041810A1 (en) * | 1995-06-08 | 1996-12-27 | Progen Industries Limited | Method and apparatus for dna extraction |
| CN104152438A (en) * | 2014-08-15 | 2014-11-19 | 四川农业大学 | Cell lysate used for extracting poultry DNA, and kit and method thereof |
| CN113528507A (en) * | 2021-07-12 | 2021-10-22 | 中国农业科学院作物科学研究所 | Kit for extracting chicken blood genome DNA by high-throughput rapid paramagnetic particle method and extraction method |
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| WO1996041810A1 (en) * | 1995-06-08 | 1996-12-27 | Progen Industries Limited | Method and apparatus for dna extraction |
| CN104152438A (en) * | 2014-08-15 | 2014-11-19 | 四川农业大学 | Cell lysate used for extracting poultry DNA, and kit and method thereof |
| CN113528507A (en) * | 2021-07-12 | 2021-10-22 | 中国农业科学院作物科学研究所 | Kit for extracting chicken blood genome DNA by high-throughput rapid paramagnetic particle method and extraction method |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117230060A (en) * | 2023-11-13 | 2023-12-15 | 苏州为度生物技术有限公司 | Pre-lysis solution, kit and method for extracting genome DNA |
| CN117230060B (en) * | 2023-11-13 | 2024-02-09 | 苏州为度生物技术有限公司 | Pre-lysis solution, kit and method for extracting genome DNA |
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