CN111607636A - A method for extracting high-quality DNA from cells of Heteroflexus alba - Google Patents

Abstract

The invention discloses a method for extracting high-quality DNA from heterocurvulus akashiwo cells, which comprises the following steps: culturing algae cells; screening algae cell lysis buffer solution; the collection method of the algae cells is groped; the method mainly comprises the steps of collecting heterocurvula akashiwo cells by using a filter membrane and selecting a proper CTAB cell lysate, and the result shows that the genomic DNA of the heterocurvula akashiwo extracted by using the novel method is superior to other methods in quality and integrity, and is suitable for PCR analysis and DNA high-throughput sequencing.

Description

A method for extracting high-quality DNA from cells of Heteroflexus alba

technical field

The invention relates to the field of DNA extraction of Heteroflexus alba, in particular to a method for extracting high-quality DNA from cells of Heteroflexus alba.

Background technique

Harmful algal blooms form as a result of certain algal blooms that can cause significant damage to marine ecosystems and local economies. The frequency, intensity and geographic distribution of HABs have increased around the world in recent years. Heterocurbita albicans is a typical red tide species, a ubiquitous and basal organism, distributed in many sea areas of the world. This algae has been widely studied because it produces fish toxins when red tides form, severely damaging marine ecosystems and killing large numbers of farmed fish. However, the mechanisms of algal bloom formation and toxin production remain unclear, to better understand how H. algal blooms form, how algal blooms kill fish, the molecular mechanisms or the biochemistry that regulates the growth and metabolism of this species Process information is very important.

DNA-based molecular biology methods are one of the effective methods to study biological characteristics. For molecular analysis, genetic engineering, such as cloning and DNA sequencing, the initial step in all of these steps is DNA extraction. There are many DNA extraction methods, such as sodium dodecyl sulfate (SDS) method, cetyltrimethylammonium bromide (CTAB) method and commercial DNA extraction kits. However, some of the DNA extraction methods described above may work for some specific organisms but not for specific algal species. The genome of H. alba is large, and it is difficult to obtain complete genomic DNA. Therefore, obtaining a high-concentration, high-purity and high-integrity genome of H. alba is a prerequisite for high-throughput DNA sequencing technology of this species.

The difficulty in extracting DNA from plants and algae is that they have unique cellular structures. The cells of Heteroflexus alba are fragile and have no cell walls. Therefore, when algal cells are collected by centrifugation, the cells aggregate to form cell clusters, making complete lysis of the algal cells more difficult. Although homogenizers can disrupt the structure of cells, physical manipulation can reduce the integrity of genomic DNA, making the DNA obtained by this method unsuitable for downstream experiments such as genome sequencing.

SUMMARY OF THE INVENTION

The purpose of the present invention is to address the defects of the prior art, and propose a method for extracting high-quality DNA from cells of Heteroflexus alba, so as to solve the problems raised by the above-mentioned background technology.

To achieve the above object, the present invention provides the following technical solutions: a method for extracting high-quality DNA from Heteroflexus alba cells, comprising the following steps:

S1: Cultivation of algal cells: the seawater used to prepare the algal culture solution is filtered through a 0.22 μm filter membrane, and sterilized by autoclaving, with a salinity of 30 PSU;

S2: Screening of algal cell lysis buffer: high-quality DNA was obtained by SDS treatment and CTAB extraction; first, algal cells were obtained from the medium, and then the cell pellets were suspended in SDS and CTAB cell lysates, respectively;

S3: Harvest algal cells: use a 0.3 μm PVDF filter membrane, then transfer the filter membrane to 0.8 mL of CTAB cell lysate (2% CTAB, 100 mM Tris-HCl pH 8.0, 1.4 M NaCl, 0.2% β-thiol) pre-warmed at 60 °C ethanol, 0.1 mg/ml proteinase K), cut the filter membrane into pieces with scissors, seal the mouth of the centrifuge tube with parafilm, incubate at 60°C for 2 hours, and gently invert and mix occasionally. Transfer the cell lysate to a new centrifuge tube;

S4: Add 800 μL of chloroform to the cell solution, invert the centrifuge tube gently for 2 minutes to mix it, stand at room temperature for 2 minutes, and then centrifuge at 4°C and 12000rpm for 10 minutes;

S5: transfer the upper aqueous phase to a new centrifuge tube, add RNase, and incubate at 37°C for 30 minutes;

S6: add equal volume of chloroform to the mixed solution after the warm bath, and centrifuge at 12,000 rpm for 10 minutes;

S7: transfer the upper aqueous phase to another new centrifuge tube, add 1/10 volume of sodium acetate and 2/3 volume of isopropanol, gently invert and mix;

S8: at room temperature, stand for 10 minutes, 4° C., 12000 rpm, and centrifuge for 5 minutes. Remove the supernatant, wash twice with 70% anhydrous ethanol, aspirate the anhydrous ethanol, and air dry at room temperature; finally, resuspend the dried DNA sample in 100 μL sterile water, and use 0.6% agarose gel for electrophoresis;

S9: Analysis of DNA Concentration, Purity and Integrity: DNA concentration was quantitatively determined using an ultra-micro spectrophotometer.

As a preferred technical solution of the present invention, the medium for culturing algal cells in the S1 is f/2 medium without adding silicon element, cultured at 20°C, and the algal cell concentration is counted using Sedgwick-Rafter Plates were counted under a microscope and cell concentrations were calculated.

As a preferred technical solution of the present invention, the calculation formula of the algal cell concentration is: _CA = DR*G*50, wherein _CA represents the algal cell concentration; DR represents the dilution ratio of the algal culture solution when counting algae; G Indicates the average number of algal cells.

As a preferred technical solution of the present invention, the specific steps of using SDS to lyse algal cells in the S2:

S2-1-1: algal cells were collected by centrifugation, the cell pellets were suspended in SDS cell lysate (0.1 MEDTA, 1% SDS), and the cell suspension was incubated at 56°C overnight;

S2-1-2: Add an equal volume of chloroform to the cell suspension, invert and mix repeatedly, centrifuge at 4°C, 12,000 rpm for 10 minutes, carefully transfer the upper aqueous phase to a new centrifuge tube, add 1/ 10 volumes of NaAc and 2/3 volumes of isopropanol;

S2-1-3: The mixed cell suspension was allowed to stand at room temperature for 10 minutes, then centrifuged at 4°C, 12,000 rpm for 5 minutes to collect DNA. The DNA was washed twice with 70% absolute ethanol and then air-dried. Dissolve DNA in sterile water.

As a preferred technical solution of the present invention, the concrete steps of using CTAB to lyse algal cells in the S2:

S2-2-1: Incubate in 0.5 ml DNA lysis buffer (10 mM Tris, pH 8.0; 100 mM EDTA, pH 8.0; 0.5% SDS and 200 μg/ml proteinase K) for 5 hours at 55°C; add 82.5 μL 5M NaCl, final concentration is 0.7M;

S2-2-2: Then add 82.5 μL of pre-warmed 10% (w/v) CTAB solution to the cell lysate, vortex for 1 minute and incubate at 56°C for 10 minutes;

S2-2-3: Add 600 μL of chloroform, 12000rpm, centrifuge for 10 minutes, transfer the supernatant to a new centrifuge tube, then add 2 volumes of DNA binding buffer to each DNA sample, the rest of the steps are the same as Zymo The DNA purification and concentration kit methods are the same.

As a preferred technical solution of the present invention, the method for determining DNA concentration in S9: according to the absorbance values at 230, 260 and 280 nm, use Nanodrop to quantitatively determine the purity of nucleic acid, and calculate the 260/280 and 260/230 The genomic DNA was extracted by the optimized method, its integrity was identified by 0.6% agarose gel electrophoresis, and the DNA bands were observed by gel nucleic acid staining.

Beneficial effects of the present invention: The method for extracting high-quality DNA from Heteroflexus alba cells proposed by the present invention mainly uses a filter membrane to collect Heteroflexus alba cells and selects a suitable CTAB cell lysate. The results showed that the new method was superior to other methods in the concentration and quality of the extracted genomic DNA of Heteroflexus alba, and was suitable for PCR analysis and high-throughput DNA sequencing.

Description of drawings

Fig. 1 is the flow chart of the present invention;

Figure 2. Analysis of the integrity and yield of genomic DNA extracted using different methods by standard electrophoresis;

Figure 3 identifies DNA integrity by standard electrophoresis;

Figure 4 Assessment of DNA quality by standard electrophoresis.

Detailed ways

The preferred embodiments of the present invention are described in detail below, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, and the protection scope of the present invention can be more clearly defined.

Embodiment 1: Please refer to FIG. 1, the present invention provides a technical solution: a method for extracting high-quality DNA from Heteroflexus alba cells: The implementation steps of the present invention are as follows:

It includes the following steps:

S1: Cultivation of algal cells: the seawater used to prepare the algal culture solution is filtered through a 0.22 μm filter membrane, and sterilized by autoclaving, with a salinity of 30 PSU;

The medium for culturing algal cells was f/2 medium without added silicon element, cultured at 20°C, and the algal cell concentration was counted under a microscope using a Sedgwick-Rafter counting plate, and then the cell concentration was calculated.

The calculation formula of the algal cell concentration is: _CA =DR*G*50, wherein _CA represents the algal cell concentration; DR represents the dilution ratio of the algal culture solution when counting algae; G represents the average value of the algal cell number.

S2: Screening of algal cell lysis buffer: high-quality DNA was obtained by SDS treatment and CTAB extraction; first, algal cells were obtained from the medium, and then the cell pellets were suspended in SDS and CTAB cell lysates, respectively;

Specific steps for lysing algal cells using SDS:

S2-1-1: algal cells were collected by centrifugation, the cell pellets were suspended in SDS buffer (0.1 MEDTA, 1% SDS), and the cell suspension was incubated at 56°C overnight;

S2-1-2: Add an equal volume of chloroform to the cell lysate, gently invert and mix, and centrifuge at 4°C, 12000 rpm for 10 minutes. Carefully transfer the upper aqueous phase to a new centrifuge tube, add 1/10 volume NaAc and 2/3 volume isopropanol;

S2-1-3: The mixed cell lysate was allowed to stand for 10 minutes at room temperature, and then centrifuged at 4°C and 12000 rpm for 5 minutes to collect DNA. The DNA was washed twice with 70% absolute ethanol and air-dried before dissolving the DNA in sterile water.

Specific steps for lysing algal cells with CTAB:

S2-2-1: Incubate in 0.5 ml DNA lysis buffer (10 mM Tris, pH 8.0; 100 mM EDTA, pH 8.0; 0.5% SDS and 200 μg/ml proteinase K) for 5 hours at 55°C; add 82.5 μL 5M NaCl, final concentration is 0.7M;

S2-2-2: Then add 82.5 μL of pre-warmed 10% (w/v) CTAB solution to the lysate, vortex for 1 minute, and incubate at 56°C for 10 minutes;

S2-2-3: Add 600 μL of chloroform, 12000rpm, centrifuge for 10 minutes, transfer the upper aqueous phase to a new centrifuge tube, then add 2 volumes of DNA binding buffer to each DNA sample, and the rest of the steps are the same as Zymo The DNA purification and concentration kit methods are the same.

S3: Harvest algal cells: use a 0.3 μm PVDF filter, then transfer the filter to 0.8 mL of CTAB buffer (2% CTAB, 100 mM Tris-HCl pH 8.0, 1.4 M NaCl, 0.2% β-mercaptoethanol) pre-warmed at 60°C , 0.1mg/ml proteinase K), cut the filter membrane with scissors, seal the bottle cap with parafilm, incubate at 60°C for 2 hours, gently invert and mix occasionally, after centrifuging the sample at 12000rpm for 15s, remove the cell lysate Transfer to a new centrifuge tube;

S4: Add 800 μL of chloroform to the cell lysate, invert the centrifuge tube gently for 2 minutes to mix it, let it stand for 2 minutes at room temperature, and then centrifuge at 4°C and 12,000 rpm for 10 minutes;

S5: transfer the upper aqueous phase to a new centrifuge tube, add RNase, and incubate at 37°C for 30 minutes;

S6: add an equal volume of chloroform to the cell lysate after the warm bath, and centrifuge at 12,000 rpm for 10 minutes;

S7: transfer the upper aqueous phase to another new centrifuge tube, add 1/10 volume of sodium acetate and 2/3 volume of isopropanol, and gently invert and mix;

S8: at room temperature, let stand for 10 minutes, 4° C., 12000 rpm, centrifuge for 5 minutes, and remove the supernatant. Wash the precipitate twice with 70% absolute ethanol, remove the absolute ethanol, and dry at room temperature; finally, resuspend the dried DNA sample in 100 μL of sterile water, and perform electrophoresis on 0.6% agarose gel;

S9: Analysis of DNA Concentration, Purity, and Integrity: DNA concentration was quantified using an ultra-micro spectrophotometer. According to the absorbance values at 230, 260 and 280 nm, the purity of nucleic acid was quantitatively determined with Nanodrop, and the values of 260/280 and 260/230 were calculated. The optimized method was used to extract the genomic DNA of Heteroflexus alba, which was purified by 0.6% agar. Its integrity was identified by glycogel electrophoresis, and DNA bands were observed by gel nucleic acid staining.

Outcome 1: Integrity of DNA

As shown in Figure 2, the genomic DNA of Heteroflexus akataide was obtained by SDS method and SDS method, followed by DNA purification with elution column and the routine DNA extraction method commonly used in laboratories, and the results of gel electrophoresis were analyzed. . DNA extracted from Heteroflexus alba by SDS method showed a strong band (Fig. 2, band 1). When DNA was extracted by SDS method and purified with Zymo purification kit, a very weak band was found (Figure 2, band 2). The results of agarose gel electrophoresis showed that DNA obtained using standard DNA extraction methods had a diffuse band after electrophoresis (FIG. 2, band 3). By analyzing the values of 260/280, 260/230 and the integrity of the DNA extracted by the three methods, it was found that the purity of the DNA was not high or the integrity was low. In addition, the collection method of algal cells also affects the concentration of DNA. The algal cells collected by the filter method showed a strong band (Fig. 4, band 1), indicating that the concentration of extracted DNA was high, and the use of magnetic beads to The cells collected by centrifugation were disrupted, and the electrophoresis results of the extracted DNA showed that the DNA bands were dispersed (Fig. 4, band 2), indicating that the integrity of the extracted DNA was low. The quality of DNA extracted by CTAB method and SDS method was compared, and it was shown that DNA extraction by CTAB method could reduce the pollution of protein.

Result 2: Purity of DNA

The nucleic acid purity is estimated by calculating the values of 260/280 and 260/230. The value of 260/280 can be used as an indicator of whether there is protein contamination. Similarly, the ratio of 260/230 can be used to monitor other contaminants such as carbohydrates, thiocyanate Guanidine acids and phenols. The 260/280 value of DNA extracted by SDS method was 1.66, and the value of 260/230 was 0.94 (Table 1, Lane 1); when the isolated DNA was further purified with a DNA purification column, the ratios of 260/280 and 260/230 were increased, respectively to 1.75 and 1.59 (Table 1, Lane 2). In comparison, the 260/280 and 260/230 values for DNA extracted using the standard method were 1.83 and 2.03, respectively (Table 1, Lane 3). Compared with the 260/280 and 260/230 (Table 2, Line 3, 4) values of the DNA extracted by the SDS method, the 260/280 and 260/230 values of the DNA extracted by the CTAB method were better (Table 2, Line 1 ,2). In order to further verify that different cell lysates obviously affect the quality of the extracted DNA, two replicates were set for SDS and CTAB lysates, and the results are shown in Figure 3. 3). Finally, the effect of cell collection method on DNA purity was investigated, and the results showed that DNA extraction from algal cells collected by the filter method resulted in higher quality genomic DNA (Fig. 4, lanes 5 and 6).

Result analysis:

The extraction of nucleic acid is the basis of genetic and molecular biology experiments of marine microalgae. For specific harmful algae such as H. alba, traditional nucleic acid extraction methods cannot obtain high-quality nucleic acids, which is not conducive to a better and comprehensive understanding of the biological characteristics of H. alba at the molecular level. Therefore, a fast, simple and reliable nucleic acid extraction method is very necessary.

Find a suitable lysis method and ideal lysis buffer for H. akashio cells. Select SDS lysate and CTAB lysate to lyse algal cells under incubation conditions at 65°C. High concentrations of DNA were obtained using the SDS method (Figure 2, Lane 1), but the ratios of 260/280 and 260/230 values were 1.66 and 0.94, respectively (Table 2, Lane 1), indicating protein and phenol contamination, which will affect the efficiency of downstream experiments. It is worth noting that although using SDS method for DNA extraction followed by DNA purification using a DNA purification column can improve the purity of the obtained DNA (Table 1, Lane 2), this process is very time-consuming, will greatly reduce the yield of DNA, and the complete low degree. It shows that SDS lysate is not a suitable lysate for extracting high-quality DNA from Heteroflexus alba.

The quality of DNA extracted by CTAB method and SDS method was compared, and it was found that different lysates had obvious influence on the quality of DNA of Heteroflexus alba. According to Figure 3 and Table 2, it can be concluded that when DNA is extracted by CTAB method, protein contamination can be greatly reduced (Figure 3, CTAB method: bands 1 and 2; SDS method: bands 3 and 4). The comparison of 260/280 and 160/230 of DNA extracted by SDS method and CTAB method in Table 2 shows that the quality of DNA extracted by CTAB method is better than that of SDS method.

The effect of cell collection method on DNA extraction results was further verified. The results are shown in Figure 4. The use of filter membranes to collect algal cells resulted in high concentrations of DNA. The algal cells were collected by centrifugation and then broken with ceramic beads, which would break the DNA into small fragments, affecting the integrity and quality of the DNA (Figure 4, band 2). This is because H. akata is a microalgae without a cell wall, surrounded by a periplasmic membrane, and centrifugation will cause the cells to clump together into a cell mass that will prevent the lysate from fully contacting the cells. Therefore, harvesting cells by centrifugation can reduce the quality of DNA extraction. The method of using ceramic beads to break up the cells after centrifugation can break up the cell mass and destroy the integrity of the DNA. In contrast, the use of filters to collect algal cells can effectively avoid the above problems. However, a small amount of RNA could still be detected in the extraction results (Figure 4, Lanes 4 and 5; Table 2, Lanes 4 and 5), and RNase could be added to remove RNA contamination without affecting the yield and integrity of DNA (Figure 4, Lanes 4 and 5). Belt 5, 6; Lane 5, 6).

In conclusion, our optimized method can obtain higher quality DNA, and CTAB method is a suitable cell lysate for obtaining high quality DNA. However, to further improve DNA quality, algal cells can be collected using a filter method, which not only has high yields, but also has good integrity, high purity, and guaranteed quality and quantity. In addition, the isolated genomic DNA is suitable for downstream processes such as PCR and high-throughput sequencing.

Table 1 Absorbance values of DNA extracted by different methods

(Fig. 2. Band 1: DNA was extracted by SDS method; Band 2: After DNA was extracted by SDS method, the DNA was purified by Zymo purification column; Band 3: DNA was extracted by conventional method; M: DNA marker).

Table 2 Absorbance values of DNA extracted by different methods

(Figure 3. Bands 1 and 2: represent DNA extracted by CTAB method; Bands 3 and 4: represent DNA extracted by SDS method; M: The molecular weight of DNAmarker is the same as in Figure 2)

Table 3 Absorbance values of DNA extracted by different methods

(Fig. 4. Band 1: algal cells were collected by filter method; band 2: algal cells were collected by centrifugation, and the cells were broken with ceramic beads; bands 3, 4: algal cells were collected by filter and then treated with CTAB buffer Lysate algal cells; Bands 5 and 6: the same as Bands 3 and 4, with the addition of RNase; M: the molecular weight of DNAMarker is the same as in Figure 2).

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention.

Claims (6)

1. A method for extracting high-quality DNA from heterocurvulus akashiwo cells is characterized by comprising the following steps:

s1 culture of algae cells: filtering seawater for preparing algae culture solution with 0.22 μm filter membrane, sterilizing at high temperature and high pressure, and salinity of 30 PSU;

s2 screening of algal cell lysis buffer: obtaining high-quality DNA by an SDS treatment method and a CTAB extraction method; firstly, obtaining algae cells from a culture solution, and then suspending cell particles in SDS and CTAB cell lysis solutions respectively;

s3, collecting algae cells: using a 0.3-micron PVDF filter membrane, transferring the filter membrane into 0.8mL CTAB buffer solution preheated at 60 ℃, shearing the filter membrane by using scissors, sealing the opening of a centrifuge tube by using a sealing membrane, incubating at 60 ℃ for 2 hours, slightly inverting and mixing from time to time, centrifuging the sample at 12000rpm for 15s, and transferring cell lysate into a new centrifuge tube;

s4, adding 800 mu L of chloroform into the cell lysate, slightly inverting the centrifuge tube for 2 minutes to mix the mixture evenly, standing the mixture for 2 minutes at room temperature, and then centrifuging the mixture for 10 minutes at 12000rpm at 4 ℃;

s5, transferring the upper aqueous phase into a new centrifuge tube, adding RNase, and carrying out warm bath at 37 ℃ for 30 minutes;

s6, adding equal volume of chloroform into the mixed solution after warm bath, and centrifuging at 12000rpm for 10 minutes;

s7, transferring the upper aqueous phase into another new centrifuge tube, adding 1/10 volumes of sodium acetate and 2/3 volumes of isopropanol, and slightly reversing and mixing;

s8, standing at room temperature for 10 minutes, standing at 4 ℃, 12000rpm, and centrifuging for 5 minutes. Removing supernatant, washing with 70% anhydrous ethanol for 2 times, sucking off anhydrous ethanol, and air drying at room temperature; finally, the dried DNA sample was resuspended in 100. mu.L of sterile water and subjected to electrophoresis using 0.6% agarose gel;

s9 analysis of DNA concentration, purity and integrity: the DNA concentration was quantitatively determined by means of an ultramicro spectrophotometer.

2. The method of claim 1, wherein the DNA is extracted from Heterocurus akashiwo cells in high quality: the culture medium for culturing the algae cells in the S1 is f/2 culture medium without adding silicon element, the algae cells are cultured under the culture condition of 20 ℃, the concentration of the algae cells is counted under a microscope by using a Sedgwick-Rafter counting plate, and then the concentration of the cells is calculated.

3. The method of claim 2, wherein the DNA is extracted from the Heterocurus akashiwo cells in high quality: the calculation formula of the algae cell concentration is as follows: c_ADR G50, wherein C_AIndicating the concentration of the algae cells; DR represents the dilution factor of the algae culture solution during the algae cultivation; g represents the average value of the number of algal cells.

4. The method of claim 1, wherein the DNA is extracted from Heterocurus akashiwo cells in high quality: the specific steps of lysis of algal cells by SDS in S2 are as follows:

s2-1-1: collecting algae cells by centrifugation, suspending cell particles in SDS lysate (0.1M EDTA, 1% SDS), and incubating the cell suspension at 56 ℃ overnight;

s2-1-2: an equal volume of chloroform was added to the cell lysate and mixed by gentle inversion. Centrifuging at 12000rpm for 10 minutes at 4 deg.C, carefully transferring the supernatant to a new centrifuge tube, adding 1/10 volumes of NaAc and 2/3 volumes of isopropanol;

s2-1-3: the mixed cell lysate is stood for 10 minutes at room temperature, centrifuged for 5 minutes at 4 ℃ and 12000rpm, DNA is collected, washed twice with 70% absolute ethyl alcohol and dried, and then the DNA is dissolved in sterile water.

5. The method of claim 1, wherein the DNA is extracted from Heterocurus akashiwo cells in high quality: the specific steps of disrupting algal cells using CTAB in S2 are as follows:

s2-2-1: incubation in 0.5ml DNA lysis buffer (10mM Tris, pH 8.0; 100mM EDTA, pH 8.0; 0.5% SDS and 200. mu.g/ml proteinase K) at 55 ℃ for 5 hours; add 82.5. mu.L of 5M NaCl to a final concentration of 0.7M;

s2-2-2: then adding 82.5 μ L of pre-preheated 10% (w/v) CTAB solution to the mixture, vortexing for 1 minute, and incubating at 56 deg.C for 10 minutes;

s2-2-3: add 600. mu.L of chloroform at 12000rpm, centrifuge for 10 minutes, transfer the upper aqueous phase to a new centrifuge tube, and add 2 volumes of DNA buffer to each DNA sample, as in Zymo's DNA purification and concentration kit.

6. The method of claim 1, wherein the DNA is extracted from Heterocurus akashiwo cells in high quality: the method for determining the DNA concentration in S9: according to the absorbance values at 230, 260 and 280nm, the purity of the nucleic acid is quantitatively determined by using Nanodrop, the ratio of 260/280 to 260/230 is calculated, the optimized method is used for extracting the genome DNA of the Heterocurus akashiwo, the integrity of the genome DNA is identified by 0.6% agarose gel electrophoresis, and the DNA bands are observed by using a gel nucleic acid staining method.

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