阅读说明：本技术 一种新型cDNA文库的制备 (Preparation of novel cDNA library ) 是由 朱也 曾繁丽 柳曹枝 于 2021-07-14 设计创作，主要内容包括：本发明涉及cDNA文库技术领域,且公开了一种新型cDNA文库的制备,将组织碎片和变性裂解液装入容量瓶中密封,再将其放入冰水中浸泡8min后取出,之后将容量瓶放入离心机中高速匀浆,离心速率为20-40秒/次,之后取出容量管,加入乙酸钠和氯仿混合后密封,再将其放入冰水中浸泡8min后取出,之后再放入离心机中离心20-40min,之后静置沉淀通过移液管转移容量管上层液体,在容量瓶中加入核酸酶抑制剂充分溶解RNA沉淀；添加两轮消减杂交及两步PCR,使Tester中代表差异表达基因的cDNA片段得到大量扩增,同时抑制了非特异性cDNA片段的扩增,使各种消减文库的特异性得到极大地提高单链Tester cDNA的均等化过程,使高、低丰度的差异表达基因都能有效分离。(The invention relates to the technical field of cDNA libraries, and discloses a preparation method of a novel cDNA library, which comprises the steps of filling tissue fragments and denatured lysate into a volumetric flask, sealing, soaking the volumetric flask in ice water for 8min, taking out, putting the volumetric flask into a centrifuge, homogenizing at a high speed, wherein the centrifugation speed is 20-40 seconds/time, taking out a volumetric flask, adding sodium acetate and chloroform, mixing, sealing, soaking the volumetric flask in ice water for 8min, taking out, putting the volumetric flask into the centrifuge, centrifuging for 20-40min, standing and precipitating, transferring upper-layer liquid of the volumetric flask through a pipette, and adding a nuclease inhibitor into the volumetric flask to fully dissolve RNA precipitate; two rounds of subtractive hybridization and two-step PCR are added to amplify a great amount of cDNA fragments representing differentially expressed genes in the Tester, and simultaneously inhibit the amplification of non-specific cDNA fragments, so that the specificity of various subtractive libraries is greatly improved in the equalization process of single-chain Tester cDNA, and the differentially expressed genes with high and low abundance can be effectively separated.)

1. The preparation of a novel cDNA library is characterized in that the used reagent consists of the following components in parts by weight:

1000ml to 500ml of DEPC water;

100ml to 50ml of 10 percent sodium sarcosinate;

250ml to 125ml of denatured lysate;

50ml to 25ml of 2M sodium acetate;

100ml to 50ml of 3M sodium acetate;

0.5M EDTA100ml～50ml；

10X MOPS 1L～0.5ml；

4x RNA Loading buffer1000ml～500ml；

50 ml-25 ml of denatured electrophoresis gel;

250ml to 125ml of denatured electrophoresis buffer solution.

2. The method for preparing a novel cDNA library according to claim 1, comprising the steps of:

s1: preparing a centrifuge tube, a pipette, a measuring cylinder, a volumetric flask, a medicine spoon and a reagent bottle, wrapping the mortar, the pipette, the measuring cylinder, the volumetric flask, the medicine spoon and the reagent bottle with tinfoil, then putting the wrapped objects into an oven for baking for 5 hours, and then putting the objects into 0.1% diethyl pyrocarbonate water for soaking overnight;

s2: placing tissue fragments and denatured lysate into a volumetric flask, sealing, placing the volumetric flask into ice water, soaking for 8min, taking out, placing the volumetric flask into a centrifuge, homogenizing at a high speed, wherein the centrifugation speed is 20-40 seconds/time, taking out the volumetric flask, adding sodium acetate and chloroform, mixing, sealing, placing the volumetric flask into the ice water, soaking for 8min, taking out, placing the volumetric flask into the centrifuge, centrifuging for 20-40min, standing and transferring upper-layer liquid of the volumetric flask through a pipette, repeating the steps until the volumetric flask is free of ethanol smell, adding a nuclease inhibitor into the volumetric flask, fully dissolving RNA precipitate, and finally placing the volumetric flask into a refrigerator of-80 ℃ for storage;

s3: taking RNA out of a volumetric flask in S2 by a pipette, putting the RNA into a centrifugal tube, adding mildew-free sterile water, adding OBB Buffer and Oligotex Suspension with proper volume, mixing and sealing, taking out the centrifugal tube, putting the centrifugal tube into 70-degree water, heating for 3min, adding an oligodeoxynucleotide primer marked by biotin and magnetic beads coated by streptavidin into the centrifugal tube, separating the magnetic beads from a low-salt Buffer solution by a magnetic separator, adding an elution Buffer solution, and repeating the magnetic separation step to obtain an mRNA solution;

s4: adding reverse transcriptase into an mRNA solution in S3, centrifuging to obtain a first cRNA chain, taking 2ul of the first cRNA chain, adding DNA Polymerase, deoxyribonucleotide triphosphate, double distilled water, isocitrate dehydrogenase and escherichia coli DNA Ligase, mixing, reacting for 2.5 hours in an environment of 20 ℃ to obtain a second cRNA chain, sequentially adding 10mM dNTP, T4 DNA Polymerase and BSA into a second chain reaction system, inactivating, adding isometric chloroform, oscillating, centrifuging, standing the reaction system at 70 ℃ for 15 minutes to inactivate T4 DNA Ligase after the ligation reaction is finished, adding Xho 10xBuffer, BSA and double distilled water, and reacting for 1.5 hoursTime of flight Inactivating the enzyme at 65 ℃ for 10 minutes to obtain the synthesized double-stranded cRNA；

S5: preparing a carrier for cloning RNA through a plasmid and a phage vector, dephosphorylating the carrier, and then sequentially adding ddH2O, T4 ligase 10X buffer, PBK (E/X) vector, cDNA, T4 DNAligase and Total to connect the carrier and the double-stranded cRNA in an environment of 14 ℃;

s6: taking a proper amount of PCR thin-walled tube, placing the PCR thin-walled tube on ice, and adding the PCR thin-walled tube into the ice in sequence according to the following system: centrifuging and precipitating the bottom after 10xbuffer, Mgcl2, DNTP, a T3 primer, a T7 primer, Taq enzyme, double distilled water and total, placing the bottom on a PCR instrument, taking down a PCR thin-walled tube after PCR reaction reaches 4 ℃, taking 7ul PCR products, adding 3ul bromoFinland for running electrophoresis, taking pictures after 1Kb DNA ladder is put for half an hour, and observing a gel picture to detect the cRNA library.

Technical Field

The invention relates to the technical field of cDNA libraries, in particular to preparation of a novel cDNA library.

Background

The cDNA library is a collection of clones formed by ligating cDNA fragments obtained by reverse transcription of all mRNA transcribed in a certain development period of an organism to a certain vector, and has tissue or cell specificity. The cDNA library is convenient for cloning and amplifying in large quantity, and the required target gene can be screened from the cDNA library, but the traditional preparation method generally utilizes a hydroxyapatite column chromatography method to separate hybrid monomers, and the technology has the defects of complex operation, long period, large nucleic acid loss and the like.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art, the invention provides preparation of a novel cDNA library, has the advantages of simplifying experimental procedures, reducing the loss of hybrids and the like, and solves the problems of complex operation, long period and large loss of nucleic acid.

(II) technical scheme

In order to achieve the purpose, the invention provides the following technical scheme: the preparation of a novel cDNA library uses the following reagents in parts by weight:

1000ml of DEPC water;

100ml of 10% sodium sarcosinate;

250ml of denaturation lysis solution;

50ml of 2M sodium acetate;

100ml of 3M sodium acetate;

0.5M EDTA100ml；

10X MOPS 1L；

4x RNA Loading buffer1000ml；

50ml of denatured electrophoresis gel;

250ml of denatured electrophoresis buffer.

According to the preparation of the novel cDNA library, the preparation method of the novel cDNA library is provided, which comprises the following steps:

s1: preparing a centrifuge tube, a pipette, a measuring cylinder, a volumetric flask, a medicine spoon and a reagent bottle, wrapping the mortar, the pipette, the measuring cylinder, the volumetric flask, the medicine spoon and the reagent bottle with tinfoil, then putting the wrapped objects into an oven for baking for 5 hours, and then putting the objects into 0.1% diethyl pyrocarbonate water for soaking overnight;

s2: placing tissue fragments and denatured lysate into a volumetric flask, sealing, placing the volumetric flask into ice water, soaking for 8min, taking out, placing the volumetric flask into a centrifuge, homogenizing at a high speed, wherein the centrifugation speed is 20-40 seconds/time, taking out the volumetric flask, adding sodium acetate and chloroform, mixing, sealing, placing the volumetric flask into the ice water, soaking for 8min, taking out, placing the volumetric flask into the centrifuge, centrifuging for 20-40min, standing and transferring upper-layer liquid of the volumetric flask through a pipette, repeating the steps until the volumetric flask is free of ethanol smell, adding a nuclease inhibitor into the volumetric flask, fully dissolving RNA precipitate, and finally placing the volumetric flask into a refrigerator of-80 ℃ for storage;

s3: taking RNA out of a volumetric flask in S2 by a pipette, putting the RNA into a centrifugal tube, adding mildew-free sterile water, adding OBB Buffer and Oligotex Suspension with proper volume, mixing and sealing, taking out the centrifugal tube, putting the centrifugal tube into 70-degree water, heating for 3min, adding an oligodeoxynucleotide primer marked by biotin and magnetic beads coated by streptavidin into the centrifugal tube, separating the magnetic beads from a low-salt Buffer solution by a magnetic separator, adding an elution Buffer solution, and repeating the magnetic separation step to obtain an mRNA solution;

s4: adding reverse transcriptase into an mRNA solution in S3, centrifuging to obtain a first cRNA chain, taking 2ul of the first cRNA chain, adding DNA Polymerase, deoxyribonucleotide triphosphate, double distilled water, isocitrate dehydrogenase and escherichia coli DNA Ligase, mixing, reacting for 2.5 hours in an environment of 20 ℃ to obtain a second cRNA chain, sequentially adding 10mM dNTP, T4 DNA Polymerase and BSA into a second chain reaction system, inactivating, adding isometric chloroform, oscillating, centrifuging, standing the reaction system at 70 ℃ for 15 minutes to inactivate T4 DNA Ligase after the ligation reaction is finished, adding Xho 10xBuffer, BSA and double distilled water, and reacting for 1.5 hoursInactivating the enzyme at 65 ℃ for 10 minutes to obtain the synthesized double-stranded cRNA；

S5: preparing a carrier for cloning RNA through a plasmid and a phage vector, dephosphorylating the carrier, and then sequentially adding ddH2O, T4 ligase 10X buffer, PBK (E/X) vector, cDNA, T4 DNA ligase and Total to connect the carrier and the double-stranded cRNA in an environment at 14 ℃;

s6: taking a proper amount of PCR thin-walled tube, placing the PCR thin-walled tube on ice, and adding the PCR thin-walled tube into the ice in sequence according to the following system: centrifuging and precipitating the bottom after 10xbuffer, Mgcl2, DNTP, a T3 primer, a T7 primer, Taq enzyme, double distilled water and total, placing the bottom on a PCR instrument, taking down a PCR thin-walled tube after PCR reaction reaches 4 ℃, taking 7ul PCR products, adding 3ul bromoFinland for running electrophoresis, taking pictures after 1Kb DNA ladder is put for half an hour, and observing a gel picture to detect the cRNA library.

(III) advantageous effects

Compared with the prior art, the invention provides preparation of a novel cDNA library, and the preparation method has the following beneficial effects:

1. according to the preparation of the novel cDNA library, a great deal of cDNA fragments representing differential expression genes in a Tester are amplified by adding two rounds of subtractive hybridization and two-step PCR, and meanwhile, the amplification of non-specific cDNA fragments is inhibited, so that the specificity of various subtractive libraries is greatly improved in the equalization process of single-chain Tester cDNA, and the differential expression genes with high and low abundance can be effectively separated.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. 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.

The invention is further described below by way of examples:

the first embodiment is as follows:

the preparation of a novel cDNA library uses the following reagents in parts by weight: DEPC water, 10% sodium sarcosinate, denatured lysate, 2M sodium acetate, 3M sodium acetate, 0.5M EDTA, 10X MOPS, 4X RNA Loading buffer1, denatured electrophoresis gel and denatured electrophoresis buffer.

According to the preparation of the novel cDNA library, firstly, a preparation method of the novel cDNA library is provided, which comprises the following steps:

s1: preparing the following reagents in parts by weight: 1000ml of DEPC water, 100ml of 10% sodium sarcosinate, 250ml of denatured lysate, 50ml of 2M sodium acetate, 100ml of 3M sodium acetate, 100ml of 0.5M EDTA, 10X MOPS 1L, 1000ml of 4X RNA Loading buffer, 50ml of denatured electrophoresis gel and 250ml of denatured electrophoresis buffer;

s2: preparing a centrifuge tube, a pipette, a measuring cylinder, a volumetric flask, a medicine spoon and a reagent bottle, wrapping the mortar, the pipette, the measuring cylinder, the volumetric flask, the medicine spoon and the reagent bottle with tinfoil, then putting the wrapped objects into an oven for baking for 5 hours, and then putting the objects into 0.1% diethyl pyrocarbonate water for soaking overnight;

s3: placing tissue fragments and denatured lysate into a volumetric flask, sealing, placing the volumetric flask into ice water, soaking for 8min, taking out, placing the volumetric flask into a centrifuge, homogenizing at a high speed, wherein the centrifugation speed is 20-40 seconds/time, taking out the volumetric flask, adding sodium acetate and chloroform, mixing, sealing, placing the volumetric flask into the ice water, soaking for 8min, taking out, placing the volumetric flask into the centrifuge, centrifuging for 20-40min, standing and transferring upper-layer liquid of the volumetric flask through a pipette, repeating the steps until the volumetric flask is free of ethanol smell, adding a nuclease inhibitor into the volumetric flask, fully dissolving RNA precipitate, and finally placing the volumetric flask into a refrigerator of-80 ℃ for storage;

s4: taking RNA out of a volumetric flask in S2 by a pipette, putting the RNA into a centrifugal tube, adding mildew-free sterile water, adding OBB Buffer and Oligotex Suspension with proper volume, mixing and sealing, taking out the centrifugal tube, putting the centrifugal tube into 70-degree water, heating for 3min, adding an oligodeoxynucleotide primer marked by biotin and magnetic beads coated by streptavidin into the centrifugal tube, separating the magnetic beads from a low-salt Buffer solution by a magnetic separator, adding an elution Buffer solution, and repeating the magnetic separation step to obtain an mRNA solution;

s5: adding reverse transcriptase into an mRNA solution in S3, centrifuging to obtain a first cRNA chain, taking 2ul of the first cRNA chain, adding DNA Polymerase, deoxyribonucleotide triphosphate, double distilled water, isocitrate dehydrogenase and escherichia coli DNA Ligase, mixing, reacting for 2.5 hours in an environment of 20 ℃ to obtain a second cRNA chain, sequentially adding 10mM dNTP, T4 DNA Polymerase and BSA into a second chain reaction system, inactivating, adding isometric chloroform, oscillating, centrifuging, standing the reaction system at 70 ℃ for 15 minutes to inactivate T4 DNA Ligase after the ligation reaction is finished, adding Xho 10xBuffer, BSA and double distilled water, and reacting for 1.5 hoursInactivating the enzyme at 65 ℃ for 10 minutes to obtain the synthesized double-stranded cRNA；

S6: preparing a carrier for cloning RNA through a plasmid and a phage vector, dephosphorylating the carrier, and then sequentially adding ddH2O, T4 ligase 10X buffer, PBK (E/X) vector, cDNA, T4 DNA ligase and Total to connect the carrier and the double-stranded cRNA in an environment at 14 ℃;

s7: taking a proper amount of PCR thin-walled tube, placing the PCR thin-walled tube on ice, and adding the PCR thin-walled tube into the ice in sequence according to the following system: centrifuging and precipitating the bottom after 10xbuffer, Mgcl2, DNTP, a T3 primer, a T7 primer, Taq enzyme, double distilled water and total, placing the bottom on a PCR instrument, taking down a PCR thin-walled tube after PCR reaction reaches 4 ℃, taking 7ul PCR products, adding 3ul bromoFinland for running electrophoresis, taking pictures after 1Kb DNA ladder is put for half an hour, and observing a gel picture to detect the cRNA library.

Example two:

the preparation of a novel cDNA library uses the following reagents in parts by weight: DEPC water, 10% sodium sarcosinate, denatured lysate, 2M sodium acetate, 3M sodium acetate, 0.5M EDTA, 10X MOPS, 4X RNA Loading buffer1, denatured electrophoresis gel and denatured electrophoresis buffer.

According to the preparation of the novel cDNA library, firstly, a preparation method of the novel cDNA library is provided, which comprises the following steps:

s1: preparing the following reagents in parts by weight: 500ml of DEPC water, 50ml of 10% sodium sarcosinate, 125ml of denatured lysate, 25ml of 2M sodium acetate, 50ml of 3M sodium acetate, 0.5M EDTA20ml, 0.5L of 10X MOPS, 500ml of 4X RNA Loading buffer, 25ml of denatured electrophoresis gel and 125ml of denatured electrophoresis buffer;

s2: preparing a centrifuge tube, a pipette, a measuring cylinder, a volumetric flask, a medicine spoon and a reagent bottle, wrapping the mortar, the pipette, the measuring cylinder, the volumetric flask, the medicine spoon and the reagent bottle with tinfoil, then putting the wrapped objects into an oven for baking for 5 hours, and then putting the objects into 0.1% diethyl pyrocarbonate water for soaking overnight;

s3: placing tissue fragments and denatured lysate into a volumetric flask, sealing, placing the volumetric flask into ice water, soaking for 8min, taking out, placing the volumetric flask into a centrifuge, homogenizing at a high speed, wherein the centrifugation speed is 20-40 seconds/time, taking out the volumetric flask, adding sodium acetate and chloroform, mixing, sealing, placing the volumetric flask into the ice water, soaking for 8min, taking out, placing the volumetric flask into the centrifuge, centrifuging for 20-40min, standing and transferring upper-layer liquid of the volumetric flask through a pipette, repeating the steps until the volumetric flask is free of ethanol smell, adding a nuclease inhibitor into the volumetric flask, fully dissolving RNA precipitate, and finally placing the volumetric flask into a refrigerator of-80 ℃ for storage;

s4: taking RNA out of a volumetric flask in S2 by a pipette, putting the RNA into a centrifugal tube, adding mildew-free sterile water, adding OBB Buffer and Oligotex Suspension with proper volume, mixing and sealing, taking out the centrifugal tube, putting the centrifugal tube into 70-degree water, heating for 3min, adding an oligodeoxynucleotide primer marked by biotin and magnetic beads coated by streptavidin into the centrifugal tube, separating the magnetic beads from a low-salt Buffer solution by a magnetic separator, adding an elution Buffer solution, and repeating the magnetic separation step to obtain an mRNA solution;

s5: adding reverse transcriptase into an mRNA solution in S3, centrifuging to obtain a first cRNA chain, taking 2ul of the first cRNA chain, adding DNA Polymerase, deoxyribonucleotide triphosphate, double distilled water, isocitrate dehydrogenase and escherichia coli DNA Ligase, mixing, reacting for 2.5 hours in an environment of 20 ℃ to obtain a second cRNA chain, sequentially adding 10mM dNTP, T4 DNA Polymerase and BSA into a second chain reaction system, inactivating, adding isometric chloroform, oscillating, centrifuging, standing the reaction system at 70 ℃ for 15 minutes to inactivate T4 DNA Ligase after the ligation reaction is finished, adding Xho 10xBuffer, BSA and double distilled water, and reacting for 1.5 hoursInactivating the enzyme at 65 ℃ for 10 minutes to obtain the synthesized double-stranded cRNA；

S6: preparing a carrier for cloning RNA through a plasmid and a phage vector, dephosphorylating the carrier, and then sequentially adding ddH2O, T4 ligase 10X buffer, PBK (E/X) vector, cDNA, T4 DNA ligase and Total to connect the carrier and the double-stranded cRNA in an environment at 14 ℃;

s7: taking a proper amount of PCR thin-walled tube, placing the PCR thin-walled tube on ice, and adding the PCR thin-walled tube into the ice in sequence according to the following system: centrifuging and precipitating the bottom after 10xbuffer, Mgcl2, DNTP, a T3 primer, a T7 primer, Taq enzyme, double distilled water and total, placing the bottom on a PCR instrument, taking down a PCR thin-walled tube after PCR reaction reaches 4 ℃, taking 7ul PCR products, adding 3ul bromoFinland for running electrophoresis, taking pictures after 1Kb DNA ladder is put for half an hour, and observing a gel picture to detect the cRNA library.

Example three:

the preparation of a novel cDNA library uses the following reagents in parts by weight: DEPC water, 10% sodium sarcosinate, denatured lysate, 2M sodium acetate, 3M sodium acetate, 0.5M EDTA, 10X MOPS, 4X RNA Loading buffer1, denatured electrophoresis gel and denatured electrophoresis buffer.

According to the preparation of the novel cDNA library, firstly, a preparation method of the novel cDNA library is provided, which comprises the following steps:

s1: preparing the following reagents in parts by weight: 750ml of DEPC water, 75ml of 10% sodium sarcosinate, 225ml of denatured lysate, 37.5ml of 2M sodium acetate, 75ml of 3M sodium acetate, 75ml of 0.5M EDTA, 0.75L of 10X MOPS, 750ml of 4X RNA Loading buffer, 37.5ml of denatured electrophoresis gel and 175ml of denatured electrophoresis buffer;

s2: preparing a centrifuge tube, a pipette, a measuring cylinder, a volumetric flask, a medicine spoon and a reagent bottle, wrapping the mortar, the pipette, the measuring cylinder, the volumetric flask, the medicine spoon and the reagent bottle with tinfoil, then putting the wrapped objects into an oven for baking for 5 hours, and then putting the objects into 0.1% diethyl pyrocarbonate water for soaking overnight;

s3: placing tissue fragments and denatured lysate into a volumetric flask, sealing, placing the volumetric flask into ice water, soaking for 8min, taking out, placing the volumetric flask into a centrifuge, homogenizing at a high speed, wherein the centrifugation speed is 20-40 seconds/time, taking out the volumetric flask, adding sodium acetate and chloroform, mixing, sealing, placing the volumetric flask into the ice water, soaking for 8min, taking out, placing the volumetric flask into the centrifuge, centrifuging for 20-40min, standing and transferring upper-layer liquid of the volumetric flask through a pipette, repeating the steps until the volumetric flask is free of ethanol smell, adding a nuclease inhibitor into the volumetric flask, fully dissolving RNA precipitate, and finally placing the volumetric flask into a refrigerator of-80 ℃ for storage;

s4: taking RNA out of a volumetric flask in S2 by a pipette, putting the RNA into a centrifugal tube, adding mildew-free sterile water, adding OBB Buffer and Oligotex Suspension with proper volume, mixing and sealing, taking out the centrifugal tube, putting the centrifugal tube into 70-degree water, heating for 3min, adding an oligodeoxynucleotide primer marked by biotin and magnetic beads coated by streptavidin into the centrifugal tube, separating the magnetic beads from a low-salt Buffer solution by a magnetic separator, adding an elution Buffer solution, and repeating the magnetic separation step to obtain an mRNA solution;

s5: adding reverse transcriptase into an mRNA solution in S3, centrifuging to obtain a first cRNA chain, taking 2ul of the first cRNA chain, adding DNA Polymerase, deoxyribonucleotide triphosphate, double distilled water, isocitrate dehydrogenase and escherichia coli DNA Ligase, mixing, reacting for 2.5 hours in an environment of 20 ℃ to obtain a second cRNA chain, sequentially adding 10mM dNTP, T4 DNA Polymerase and BSA into a second chain reaction system, inactivating, adding isometric chloroform, oscillating, centrifuging, standing the reaction system at 70 ℃ for 15 minutes to inactivate T4 DNA Ligase after the ligation reaction is finished, adding Xho 10xBuffer, BSA and double distilled water, and reacting for 1.5 hoursInactivating the enzyme at 65 ℃ for 10 minutes to obtain the synthesized double-stranded cRNA；

S6: preparing a carrier for cloning RNA through a plasmid and a phage vector, dephosphorylating the carrier, and then sequentially adding ddH2O, T4 ligase 10X buffer, PBK (E/X) vector, cDNA, T4 DNA ligase and Total to connect the carrier and the double-stranded cRNA in an environment at 14 ℃;

s7: taking a proper amount of PCR thin-walled tube, placing the PCR thin-walled tube on ice, and adding the PCR thin-walled tube into the ice in sequence according to the following system: centrifuging and precipitating the bottom after 10xbuffer, Mgcl2, DNTP, a T3 primer, a T7 primer, Taq enzyme, double distilled water and total, placing the bottom on a PCR instrument, taking down a PCR thin-walled tube after PCR reaction reaches 4 ℃, taking 7ul PCR products, adding 3ul bromoFinland for running electrophoresis, taking pictures after 1Kb DNA ladder is put for half an hour, and observing a gel picture to detect the cRNA library.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.