CN111154752A

CN111154752A - Preparation method of PCR template DNA of yeast monoclonal colony

Info

Publication number: CN111154752A
Application number: CN202010088736.4A
Authority: CN
Inventors: 戴凌燕; 刘微; 李志江; 张东杰; 王长远
Original assignee: Heilongjiang Bayi Agricultural University
Current assignee: Heilongjiang Bayi Agricultural University
Priority date: 2020-02-12
Filing date: 2020-02-12
Publication date: 2020-05-15

Abstract

The invention provides a preparation method of PCR template DNA of yeast monoclonal colony, belonging to the technical field of molecular biology detection; the preparation method comprises the following steps: 1) adding thalli of a yeast monoclonal colony into ethyl acetate, performing vortex oscillation for 3-6 min, and heating in a water bath at 90-100 ℃ until the ethyl acetate is completely evaporated to obtain an extract; 2) mixing the extract with sterile water, carrying out vortex oscillation for 3-6 min, and heating in a water bath at 90-100 ℃ for 3-6 min to obtain the PCR template DNA of the yeast monoclonal colony. The method is simple and quick to operate, the time required for preparing the PCR template of a monoclonal colony is only 15-25 min, and compared with the time required for preparing the bacterial liquid genome DNA for 2-6 h, the method greatly shortens the experiment time, reduces the workload and reduces the experiment difficulty.

Description

Preparation method of PCR template DNA of yeast monoclonal colony

Technical Field

The invention relates to the technical field of molecular biology detection, in particular to a preparation method of PCR template DNA of yeast monoclonal colony.

Background

Yeast, the simplest eukaryote, has been an ideal model organism for studying physiological and biochemical functions, and is called Escherichia coli among eukaryotes. Because of the special metabolic pathway and the simple genetic operation method, the yeast is widely applied as a host bacterium for gene cloning and expression all the time, and is also a kind of microorganism which is applied earlier in human practice.

At present, in the process of genetic modification of yeast, the genome of the yeast is generally required to be extracted as a PCR template so as to carry out target gene amplification or positive clone screening. However, the extraction of the yeast genome takes a long time, and usually 1 to 3 days are needed to accumulate enough bacteria according to the growth cycle (100 to 300min) of the yeast; the extraction process is generally different from 2 to 6 hours, the operation process is complex, and the large-scale yeast transformant screening and target gene acquisition cannot be met.

Disclosure of Invention

The invention aims to provide a preparation method of PCR template DNA of yeast monoclonal colony, which is short in time consumption and high in efficiency.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a preparation method of PCR template DNA of yeast monoclonal colony, which comprises the following steps:

1) adding thalli of a yeast monoclonal colony into ethyl acetate, performing vortex oscillation for 3-6 min, and heating in a water bath at 90-100 ℃ until the ethyl acetate is completely evaporated to obtain an extract;

2) mixing the extract with sterile water, carrying out vortex oscillation for 3-6 min, and heating in a water bath at 90-100 ℃ for 3-6 min to obtain the yeast monoclonal colony DNA.

Preferably, the vortex oscillation time in step 1) is 5 min.

Preferably, the time of the vortex oscillation in the step 2) is 5 min.

Preferably, the time for heating in the water bath in the step 2) is 5 min.

The invention also provides the PCR template DNA of the yeast monoclonal colony prepared by the preparation method in the scheme.

The invention provides a preparation method of PCR template DNA of yeast monoclonal colony, which has the following beneficial effects:

(1) the method is simple and quick to operate, the time required for preparing the PCR template of a monoclonal colony is only 15-25 min, and compared with the time required for preparing the bacterial liquid genome DNA for 2-6 h, the method greatly shortens the experimental time, reduces the workload and reduces the experimental difficulty;

(2) in contrast to other methods, the present invention does not require the use of various enzymes or special laboratory equipment. The glusulase commonly used in the enzymolysis method is about 180 yuan/g, the cellulase is more as high as 710 yuan/g, and the reagent required for preparing a monoclonal PCR template of the invention is only 100 mu L ethyl acetate, the price is about 0.01 yuan; the consumable material only needs one centrifugal tube of 1.5mL, and the price is about 0.04 yuan; all the instruments are conventional laboratory instruments, the total price for preparing a monoclonal PCR template is not more than 0.1 yuan, so that the method has great price advantage and can effectively save the experiment cost;

(3) compared with the existing colony PCR method, the template prepared by the invention can effectively amplify the target gene, has stable effect, has a success rate of over 90 percent, and can completely meet the amplification and identification of the target gene;

(4) the invention provides a new method which is rapid, economic and effective for the amplification of target genes in yeast genomes and the large-scale screening and identification of yeast transformants, and can eliminate the worry that the extraction of genome DNA wastes time and labor and the large dosage of expensive reagents for screening transformants in a large scale.

Drawings

FIG. 1a is the colony PCR result of primer 1448, wherein M: marker (DL 2000); c: blank control (deionized water as template); 1-10: 10 monoclonals of Saccharomyces rouxii;

FIG. 1b shows the PCR result of extracting genomic DNA from the bacterial solution of primer 1448, wherein M: marker (DL 2000); c: blank control (deionized water as template); 1-10: respectively corresponding 10 zygosaccharomyces rouxii monoclonals in 1 a;

FIG. 1c is the colony PCR result of primer 1248, where M: marker (DL 2000); c: blank control (deionized water as template); 1-10: 10 monoclonals of Saccharomyces rouxii;

FIG. 1d shows the PCR result of extracting genomic DNA from the bacterial liquid with the primer 1248, where M: marker (DL 2000); c: blank control (deionized water as template); 1-10: respectively corresponding 10 zygosaccharomyces rouxii monoclonals in 1 c;

FIG. 1e is the colony PCR result for primer 944, where M: marker (DL 2000); c: blank control (deionized water as template); 1-10: 10 monoclonals of Saccharomyces rouxii;

FIG. 1f shows the PCR result of the genomic DNA extracted from the bacterial suspension with primer 944, wherein M: marker (DL 2000); c: blank control (deionized water as template); 1-10: respectively corresponding 10 zygosaccharomyces rouxii monoclonals in 1 e;

FIG. 2a is the colony PCR result of primer 754, where M: marker (DL 2000); c: blank control (deionized water as template); p: positive control (plasmid TPI-pECS-URA (MCS1) as template); 1-10: 10 monoclonals of the transformant of the zygosaccharomyces rouxii;

FIG. 2b shows the PCR result of extracting genomic DNA from the bacterial liquid of the primer 754, wherein M: marker (DL 2000); c: blank control (deionized water as template); p: positive control (plasmid TPI-pECS-URA (MCS1) as template); 1-10: the corresponding 10 transformants of Saccharomyces rouxii were each monoclonal in 2 a.

Detailed Description

Adding thalli of a yeast monoclonal colony into ethyl acetate, performing vortex oscillation for 3-6 min, and heating in a water bath at 90-100 ℃ until the ethyl acetate is completely evaporated to obtain an extract; the time of the vortex oscillation is preferably 5 min; the time for heating in the water bath is preferably 5 min.

The adding amount of the thallus of the yeast monoclonal colony is not particularly limited, and is based on the conventional setting in the field; in the specific implementation process of the invention, the bacteria of the yeast monoclonal colony are picked by the tip of the toothpick, the number of the bacteria is only visible to the naked eye, and the influence on the experimental effect caused by the introduction of the culture medium due to the picking of excessive bacteria is avoided.

In the invention, the ethyl acetate is used for dissolving the cell wall of the yeast cell and further destroying the cell wall and the biomembrane structure by combining vortex oscillation and water bath heating; the yeast cell wall can be fully digested, the biological membrane is broken and the genome DNA is released through the treatment of the invention.

After the extract is obtained, the extract is mixed with sterile water, vortex and shake are carried out for 3-6 min, and then water bath heating is carried out for 3-6 min at the temperature of 90-100 ℃, so that yeast monoclonal colony DNA is obtained; the time of the vortex oscillation is preferably 5 min; the time for heating in the water bath is preferably 5 min.

The invention also provides the PCR template DNA of the yeast monoclonal colony prepared by the preparation method in the scheme. The yeast monoclonal colony DNA prepared by the preparation method can be directly used as a template for yeast PCR reaction.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

(1) Accurately adding 100 μ L ethyl acetate into 1.5mL centrifuge tube, dipping appropriate amount of Zygosaccharomyces rouxii (China Industrial microorganism culture Collection center number: ATCC 32899) monoclonal thallus, adding into centrifuge tube, stirring thoroughly, and totally 10 clones;

(2) vortex and shake the centrifugal tube for 5 min;

(3) opening a centrifugal tube, boiling water bath for about 3min, and completely evaporating ethyl acetate;

(4) adding 50 mu L of sterile water into the centrifuge tube, and carrying out vortex oscillation for 5 min;

(5) boiling water bath for 5min again to obtain colony PCR template;

(6) amplifying by using the Dongsheng organism PCRMix, adding 2 XPCRMix 20 mu L into a PCR tube, and adopting 1448-F and 1448-R respectively; 1248-F, 1248-R; three pairs of primers (see Table 1) of 944-F and 944-R were verified, and PCR was performed with 2. mu.L of each of the upstream and downstream primers and 3. mu.L of colony PCR template, and with 50. mu.L of final PCR system by adding ultrapure water. The specific reaction conditions are as follows: 95 deg.C for 5 min; 35 cycles of 95 deg.C, 30s, 57 deg.C, 30s, 72 deg.C, 1 min; extending for 10min at 72 ℃;

(7) the PCR amplification product was detected by electrophoresis using 1% agarose gel, 1 XTAE as the electrophoresis solution, with a sample size of 5. mu.L, constant voltage of 100V, and electrophoresis for about 45 min. The results were recorded using a gel imager, see fig. 1 a-1 f.

TABLE 1 primers for Gene amplification and Positive transformant screening

Comparative example 1

(1) 10 clones selected in example 1 were cultured in YPD liquid medium for 24 hours, centrifuged at 12000rpm/min for 1min, and the cells were collected;

(2) fully grinding the thalli in liquid nitrogen to fine powder, and filling the powder into 1.5mL centrifuge tubes, wherein each tube does not exceed 100 mg;

(3) adding DNA extractive solution 600 μ L, water bathing at 60 deg.C for 30min, the components of the extractive solution are shown in Table 2;

TABLE 2 DNA extract preparation

(4) Centrifuging at 12000rpm/min and 4 deg.C for 5min, collecting supernatant, adding equal volume of chloroform, mixing, standing for 10min, and centrifuging at 12000rpm/min and 4 deg.C for 5 min;

(5) collecting supernatant, adding equal volume of isopropanol, mixing, and standing at-20 deg.C for 30 min;

(6) centrifuging at 12000rpm/min at 4 deg.C for 5min, and removing supernatant;

(7) adding 1mL of 70% ethanol, shaking for cleaning, 12000rpm/min, centrifuging at 4 deg.C for 5min, removing supernatant, opening cover, and air drying on a clean bench;

(8) adding 100 mu L of sterile water into a centrifuge tube, dissolving in a water bath at 60 ℃, shaking up to obtain genome DNA, and preserving at-20 ℃;

(9) PCR Mix of the Dongsheng organism was used for amplification, and 2 XPCR Mix20 μ L was added to the PCR tube using 1448-F and 1448-R, respectively; 1248-F, 1248-R; three pairs of primers (see Table 1) of 944-F and 944-R were verified, and PCR was performed with 2. mu.L of each of the upstream and downstream primers and 3. mu.L of the genomic DNA template, and with the addition of ultrapure water to make the final system of the PCR reaction to be 50. mu.L. The specific reaction conditions are as follows: 95 ℃ for 5 min; 35 cycles of 95 deg.C, 30s, 57 deg.C, 30s, 72 deg.C, 1 min; extending for 10min at 72 ℃;

(10) the PCR amplification product was detected by electrophoresis using 1% agarose gel, 1 XTAE as the electrophoresis solution, 5. mu.L of sample was applied to each well, constant voltage 100V, and electrophoresis was performed for about 45 min. The results were recorded using a gel imager, see fig. 1 a-1 f.

The results of the comparison of example 1 and comparative example 1 are as follows:

the colony PCR results for primer 1448 are shown in fig. 1a, where M: marker (DL 2000); c: blank control (deionized water as template); 1-10: 10 monoclonals of Saccharomyces rouxii;

the PCR results of the genomic DNA extraction from the bacterial solution of primer 1448 are shown in FIG. 1b, wherein M: marker (DL 2000); c: blank control (deionized water as template); 1-10: respectively corresponding 10 zygosaccharomyces rouxii monoclonals in 1 a;

the colony PCR results (1a) of clones No. 1-10 are completely consistent with the PCR results (FIG. 1b) with the genome as the template, and have obvious specific amplification bands.

The colony PCR results for primer 1248 are shown in fig. 1c, where M: marker (DL 2000); c: blank control (deionized water as template); 1-10: 10 monoclonals of Saccharomyces rouxii;

the PCR results of the bacterial liquid extraction of genomic DNA from primer 1248 are shown in FIG. 1d, where M: marker (DL 2000); c: blank control (deionized water as template); 1-10: respectively corresponding 10 zygosaccharomyces rouxii monoclonals in 1 c;

the colony PCR results (FIG. 1c) of clones No. 1-10 are completely consistent with the PCR results (FIG. 1d) with the genome as the template, and have obvious specific amplification bands.

The colony PCR results for primer 944 are shown in fig. 1e, where M: marker (DL 2000); c: blank control (deionized water as template); 1-10: 10 monoclonals of Saccharomyces rouxii;

the PCR results of the bacterial liquid extraction of genomic DNA with primer 944 are shown in FIG. 1f, wherein M: marker (DL 2000); c: blank control (deionized water as template); 1-10: respectively corresponding 10 zygosaccharomyces rouxii monoclonals in 1 e;

except the PCR of clone No. 3 colony, the PCR results of other 9 clones are identical to the PCR result with bacterial liquid genome as template (FIG. 1e, 1f), and there is obvious specific amplification band.

Example 2

(1) Taking a zygosaccharomyces rouxii competent cell, transferring TPI-pECS-URA (MCS1) plasmid into the zygosaccharomyces rouxii competent cell by electric shock, coating a flat plate, and culturing until macroscopic monoclonal thallus grows out for later use;

(2) accurately adding 100 mu L of ethyl acetate into a 1.5mL centrifuge tube, dipping a proper amount of zygosaccharomyces rouxii monoclonal bacteria, adding into the centrifuge tube, and fully stirring for 10 clones;

(3) vortex and shake the centrifugal tube for 5 min;

(4) opening a centrifugal tube, boiling water bath for about 3min, and completely evaporating ethyl acetate;

(5) adding 50 mu L of sterile water into the centrifuge tube, and carrying out vortex oscillation for 5 min;

(6) boiling water bath for 5min again to obtain colony PCR template of the electrically transformed bacteria;

(7) PCR amplification was performed by using PCR Mix of the Dongshan organism, adding 20. mu.L of 2 XPCR Mix to the PCR tube, verifying the PCR tube with 754-F and 754-R (see Table 1), adding 2. mu.L of each of the upstream and downstream primers, 3. mu.L of the colony PCR template of the electro-transformed bacteria, and adding ultrapure water to make the final system of the PCR reaction to be 50. mu.L. The specific reaction conditions are as follows: 95 deg.C for 5 min; 35 cycles of 95 deg.C, 30s, 57 deg.C, 30s, 72 deg.C, 1 min; extending for 10min at 72 ℃;

(8) the PCR amplification product was detected by electrophoresis using 1% agarose gel, 1 XTAE as the electrophoresis solution, 5. mu.L of sample was applied to each well, constant voltage 100V, and electrophoresis was performed for about 45 min. The results were recorded using a gel imager, see fig. 2a and 2 b.

Comparative example 2

(1) Taking bacterial liquid obtained by culturing 10 electrotransformation clones selected in example 2 with YPD liquid culture medium for 24h, respectively, centrifuging at 12000rpm/min for 1min, and collecting thallus;

(3) adding 600 μ L of the DNA extract of comparative example 1, and water bath at 60 deg.C for 30 min;

(5) collecting supernatant, adding isopropanol of the same volume, mixing, and standing at-20 deg.C for 30 min;

(6) centrifuging at 12000rpm/min at 4 deg.C for 5min, and removing supernatant;

(8) adding 100 mu L of sterile water into a centrifuge tube, dissolving in water bath at 60 ℃, shaking up to obtain genome DNA, and preserving at-20 ℃;

(9) PCR amplification was performed by using PCR Mix of the Dongshan organism, adding 20. mu.L of 2 XPCR Mix to the PCR tube, verifying the PCR using 754-F and 754-R (see Table 1), 2. mu.L each of the upstream and downstream primers, 3. mu.L of the genomic DNA template, and adding ultrapure water to make the final system of the PCR reaction 50. mu.L. The specific reaction conditions are as follows: 95 deg.C for 5 min; 95 ℃ for 30 s; 57 ℃ for 30 s; at 72 ℃ for 1min for 35 cycles; extending for 10min at 72 ℃;

(10) the PCR amplification product was detected by electrophoresis using 1% agarose gel, 1 XTAE as the electrophoresis solution, 5. mu.L of sample was applied to each well, constant voltage 100V, and electrophoresis was performed for about 45 min. The results were recorded using a gel imager, see fig. 2a and 2 b.

The results of comparing example 2 with comparative example 2 are as follows:

the colony PCR results for primer 754 are shown in fig. 2a, where M: marker (DL 2000); c: blank control (deionized water as template); p: positive control (plasmid TPI-pECS-URA (MCS1) as template); 1-10: 10 monoclonals of the transformant of the zygosaccharomyces rouxii;

the PCR results of the genomic DNA extraction from the bacterial suspension of primer 754 are shown in FIG. 2b, wherein M: marker (DL 2000); c: blank control (deionized water as template); p: positive control (plasmid TPI-pECS-URA (MCS1) as template); 1-10: respectively, 10 corresponding monoclonals of the zygosaccharomyces rouxii transformants in 2 a;

in colony PCR group of electric transformation bacteria, clone No. 2, clone No. 5 and clone No. 8 have obvious specific amplification band; in the PCR group of bacterial liquid genome, No. 2, 5, 6 and 8 clones have obvious specific amplification bands; in colony PCR and bacterial liquid genome PCR, the results of No. 2, 5 and 8 clones are completely consistent, and the clones are determined to be positive transformants.

As can be seen from the above examples and comparative examples, the DNA of the yeast monoclonal colony prepared by the preparation method of the invention can be directly used as a template for the PCR reaction of the yeast, and a target fragment on a genome is obtained by amplification, and compared with the method of extracting the genomic DNA by using a bacterial liquid as the template, the accuracy can reach more than 90%. The preparation method can realize the rapid and effective preparation of the colony PCR DNA template, and the quality of the prepared sample can realize the amplification of the target gene on the yeast genome and the screening of the yeast positive transformant. The invention provides a rapid, economic and effective new method for obtaining target genes on a genome and screening large-scale yeast transformants.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

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Claims

1. A preparation method of PCR template DNA of yeast monoclonal colony comprises the following steps:

2. The method of claim 1, wherein the vortex oscillation time in step 1) is 5 min.

3. The method as claimed in claim 1, wherein the period of the vortex oscillation in step 2) is 5 min.

4. The method of claim 1, wherein the water bath heating time in step 2) is 5 min.

5. A PCR template DNA for a monoclonal colony of yeast prepared by the method of any one of claims 1 to 4.