CN109055360A - A method of extracting anopheles total serum IgE - Google Patents
A method of extracting anopheles total serum IgE Download PDFInfo
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Abstract
The present invention relates to anopheles PNA separating and purifying technology fields, in particular to a kind of method for extracting anopheles total serum IgE, this method comprises: preparation RNA extract liquor, the coarse extraction of anopheles total serum IgE, repeatedly extracting reduces pigment and influences, butyl glycol ether precipitating centrifugation, ethanol washing, dissolving step in DEPC water, reagent price provided by the invention is cheap, laboratory is generally common, operation of the present invention step is simple, it is extracted three times, effectively remove the influence of pigment in contaminating genomic DNA and anopheles body, the RNA mass of acquisition is stablized, without protein, the pollution such as phenol, the requirement of the molecular biology experiments such as energy further satisfaction RT-PCR.
Description
Technical field
The invention belongs to anopheles RNA separating and purifying technology fields, and in particular to a method of extract anopheles total serum IgE.
Background technique
Anopheles is the primary vehicle of malaria, and malaria is to seriously endanger a kind of communicable disease of human health,
The public health problem that control is badly in need of in the whole world is classified as by WHO.Research anopheles and malaria infection propagation contacting and have between the two
Significance.It is one of the important link for carrying out anopheles molecular biology research that RNA is extracted from anopheles, to carry out Northern
(trace) hybridization analysis, purifying mRNA are to be used for In Vitro Translation or establish the downstreams such as cDNA library, RT-PCR and differential analysis point
Sub- biological study.And the success or failure for extracting experiment depend primarily on the quality of anopheles total serum IgE.The method for extracting RNA at present has different
Guanidine thiocyanate phynol method, tradition SDS method, TRIzoL method, hot borate method etc..However, a large amount of articles delivered all reflect
There is different difficulties when separating the RNA of high quality, is mainly shown as RNA vulnerable to the degradation of RNase, RNA mass by material sample
This limitation.The most small volume of anopheles, and contain a large amount of pigments, separation high quality RNA difficulty is larger.
In currently used RNA extraction method, main matter is exactly guanidinium isothiocyanate in TRIzoL reagent.Guanidinium isothiocyanate
It is strong protein denaturant, nucleoprotein and nucleic acid depolymerization can be made, and can strong inhibition RNase activity；It is tried with TRIzoL
Other compositions in agent: the interaction such as phenol, beta -mercaptoethanol can more efficiently inhibit RNase active, keep cell broken
It is bad, release RNA.The total serum IgE of TRIzoL extracting can be avoided DNA and protein contamination, but TRIzoL reagent price is expensive,
One bottle of 100mL TRIzoL (invitrogen) reagent unit price only can be used 80-100 times between 2000-5000RMB.
Traditional SDS method releases cell rupture using lauryl sodium sulfate (SDS) this strong protein denaturant
RNA, and inhibit RNase active；The water-saturated phenol of addition can also make protein denaturation, precipitating, then from nucleic acid extraction liquid
Separation of Proteins is removed；The addition of sodium acetate solution creates acid aqueous environment, makes partial nucleic acid can be by phenol extract
Absorption.And the size of the mutually adsorbed nucleic acid molecular weight of phenol and the pH value of aqueous environment are closely related.It is subsequently added into chloroform, is accelerated
The layering of organic phase and water phase, RNA enter in water phase；RNA precipitate is come out followed by isopropanol.This method utilizes phenol, chloroform
Extracting, it is at low cost, but with the anopheles RNA of this method extraction, there are obvious DNA pollution and pigmentations, and it is raw to be unsuitable for downstream molecules
Object research.
In order to simplify RNA extraction method, extraction efficiency is improved.Many companies develop a series of RNA and extract examination at this stage
Agent box, although kit extraction efficiency is high, extraction effect is good, its step repeats cumbersome mostly, and is directed to different materials, choosing
It also will appear different-effect with different kits.Such as the RNAprep pure Tissue Kit RNAprep pure of TIANGEN
Sample size handled by (centrifugal column type), which has, clearly to be limited, while needing in addition to prepare RNase-Free DNase.(TIANGEN)
The band that is obtained through agarose gel electrophoresis of anopheles RNA that DNA/RNA IsoLation Kit (centrifugal column type) is extracted it is faint and
There are small molecule 5sRNA Loss.Kit price is higher, general domestic universal 50preps total RNA extraction reagent box
Price is at 2000 yuan or so, and when needing to extract sample rna for a long time, consuming is excessive, cannot be generally applicable to vast laboratory.
Summary of the invention
According to the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to propose for disadvantage mentioned above, hair
A kind of bright economical and efficient anopheles method for extracting total RNA, this method agents useful for same laboratory is generally common, easy to operate, at low cost,
RNA is up-to-standard, and extraction effect is stablized, and is able to solve and extracts that anopheles total serum IgE is imperfect, DNA pollution is serious, there are pigment shadows
The problems such as ringing phenomenon, the RNA that can be used for high-volume anopheles sample is extracted and prolonged use.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention are as follows:
A method of extracting anopheles total serum IgE, the specific steps are as follows:
1) the hungry anopheles raised is taken, then freezing processing the anopheles after freezing is added in Buffer solution A, piping and druming
It mixes, is stored at room temperature, obtains anopheles tissue homogenate；
2) Buffer B solution is first added into obtained anopheles tissue homogenate, is successively mixed through piping and druming, incubation at room temperature,
Centrifugation 1 extracts supernatant liquor 1 into centrifuge tube, then Buffer C solution is added into supernatant liquor 1, successively mixes through piping and druming,
Incubation at room temperature, centrifugation 2 extract supernatant liquor 2 into centrifuge tube, then Buffer solution D are added into supernatant liquor 2, successively pass through
It blows and beats and mixes, incubation at room temperature, centrifugation 3 extracts supernatant liquor 3 into centrifuge tube, then addition Buffer E is molten into supernatant liquor 3
Liquid is successively mixed through piping and druming, and refrigeration is incubated for, and centrifugation 4 goes liquid to stay sediment, and it is molten that Buffer F is added in last sediment upwards
Liquid is successively mixed through piping and druming, and centrifugation 5 goes liquid to stay object, drying at room temperature 3-5min, be dissolved in 20-30 μ L DEPC water it is subzero-
It is saved at 75-85 DEG C.
Preferably, the freezing processing: temperature is subzero 15-25 DEG C, duration 3-10min.
Preferably, the Buffer solution A is full by the lauryl sodium sulfate (SDS) of 100 μ L concentration 10%, 500 μ L water
It is constituted with phenol, 200 μ L EPC water and 5 μ L beta -mercaptoethanols.
Preferably, the Buffer B solution is sodium-acetate buffer, concentration 2moL/L, pH 3.2-3.9.
Preferably, the Buffer C solution includes following component: water-saturated phenol, chloroform, isoamyl alcohol, and volume ratio is
125:24:1。
Preferably, the Buffer solution D is chloroform, and Buffer E solution is butyl glycol ether, and Buffer F solution is
Ethyl alcohol.
Preferably, the when a length of 12-18s that the piping and druming mixes.
Preferably, the revolving speed of the centrifugation 1-4 is 12000rpm/min, and temperature is 4 DEG C, time 10-15min, centrifugation 5
Revolving speed be 7500rpm/min, temperature be 4 DEG C, time 5-7min.
Preferably, the duration 3-5min of the incubation at room temperature, the temperature for refrigerating incubation is subzero 18-23 DEG C, duration 25-
35min。
Preferably, the quantity of the anopheles is 3-20.
Compared with prior art, the medicine have the advantages that
1. extraction agent formulations provided by the invention are simple, cheap, extraction efficiency is high, and RNA product is stablized.And do not make
Releasable high poisonous gas is contacted with acid with virulent property with guanidinium isothiocyanate, and is sucked, intake, skin contact
It causes to damage；Extraction reagent laboratory is common, and operating procedure is simple, at low cost, can efficiently extract anopheles total serum IgE；The party
Method is suitble to the extraction of single or more anopheles total serum IgEs simultaneously.
2. a small amount of beta -mercaptoethanol, the disulfide bond in chief destructive ribonuclease proteins, β-sulfydryl is added in the present invention
Ethyl alcohol and SDS combination preferably inhibit RNase activity, when preparing tissue homogenate, endogenous RNA enzyme may make to inactivate.
3. it is 2moL/L, sodium acetate of the pH value within the scope of 3.2-3.9 that concentration, which is added, in the present invention after homogenate mixes well
Buffer is preferably mixed with homogenate convenient for NaAc buffer；Sodium acetate solution creates acid aqueous environment simultaneously, makes part
Nucleic acid can be adsorbed by phenol extract, and the size of the mutually adsorbed nucleic acid molecular weight of phenol and the pH value of aqueous environment are closely related, because
This is verified by experimental design, and the present invention selects acid sodium acetate solution of the pH value within the scope of 3.2-3.9, is taken out in second of phenol
Total serum IgE is purified when mentioning, and genomic DNA is assigned in phenol phase and is removed, so that the anopheles total serum IgE of high quality is obtained,
The pollution such as no DNA and protein.
4. the present invention again purifies total serum IgE using chloroform, influence of the anopheles pigment to its RNA mass is reduced.
5. the present invention precipitates RNA using butyl glycol ether, sedimentation effect is good, and the sedimentation time is shorter, and RNA mass
Stablize.
Detailed description of the invention
Fig. 1 is that the anopheles total serum IgE that the method for the present invention provides mainly extracts flow chart；
Fig. 2 is the anopheles total serum IgE extracted according to 1 step of embodiment and tradition SDS method, tradition TRIzoL method, RNA isolation kit
(TIANGEN) electrophoretogram of the RNA extracted, wherein the RNA that A- tradition TRIzoL method is extracted, B-TIANGEN RNA isolation kit extract
RNA, C- tradition SDS method extract RNA, D- improved method extract RNA, M-D2000Marker.
Fig. 3 is the electrophoretogram of the product after being expanded after RT-PCR according to the anopheles total serum IgE that 1 step of embodiment is extracted,
In, the product after the anopheles total serum IgE amplification that A, B, C, D- embodiment 1 is extracted, M1-DL2000Marker.
Specific embodiment
Below by the description to embodiment, the present invention is described in further detail by specific embodiment, with side
Those skilled in the art are helped to have more complete, accurate and deep understanding to inventive concept of the invention, technical solution.
Embodiment 1
A method of extracting anopheles total serum IgE, comprising the following steps:
(1) the DEPC aqueous solution of 10%SDS is prepared, for use；
(2) preparation 2moL/L, DEPC aqueous solution, that is, Buffer B solution of pH=3.6 sodium acetate, for use；
(3) it prepares Buffer solution A: sequentially adding 200 μ L DEPC into No.1 1.5mL RNase-Free centrifuge tube
Water, 100 μ L10%SDS, 500 μ L water-saturated phenols, 5 μ L beta -mercaptoethanols, concussion is uniformly mixed up and down, and it is molten to obtain Buffer A
Liquid is placed on ice chest stand-by；
(4) it prepares Buffer C solution: sequentially adding water-saturated phenol, chlorine into No. two 1.5mL RNase-Free centrifuge tubes
Imitative and isoamyl alcohol, concussion is uniformly mixed up and down, obtains Buffer C solution, for use；Wherein, water-saturated phenol, chloroform and isoamyl alcohol
Volume ratio be 125:24:1；
(5) coarse extraction of anopheles total serum IgE: taking 3 hungry work anopheles for raising three days, is put into new RNase-Free centrifugation
Pipe is placed in -20 DEG C of refrigerator 5min, and then careful take out is placed in No.1 1.5mL RNase-Free centrifuge tube, and grinding is abundant, room
After temperature places 2min, Buffer B solution is added thereto, piping and druming mixes, it is incubated at room temperature 5min, at 4 DEG C, 12000rpm/min,
It is centrifuged 15min, supernatant liquor 1；
(6) supernatant liquor 1 after step (5) centrifugation is transferred to completely in No. three 1.5mL RNase-Free centrifuge tubes,
Isometric Buffer C solution is added thereto, piping and druming mixes, vibrates 15s after covering tightly, be stored at room temperature 5min；At 4 DEG C,
12000rpm/min is centrifuged 15min, obtains supernatant liquor 2, the supernatant liquor 2 after centrifugation is transferred to No. four 1.5mL completely
In RNase-Free centrifuge tube, isometric chloroform is added and mixes well, is stored at room temperature 3min, at 4 DEG C, 12000rpm/min,
It is centrifuged 10min, obtains supernatant liquor 3, the supernatant liquor 3 after centrifugation is transferred to No. five 1.5mL RNase-Free centrifugations completely
Isometric butyl glycol ether is added in Guan Zhong, and the centrifuge tube that turns upside down after covering tightly extremely mixes, -20 DEG C of standing 30min, at 4 DEG C,
12000rpm/min is centrifuged 10min, liquid is gone to stay sediment, is precipitated with the ethanol washing that volumetric concentration is 80%, at 4 DEG C,
7500rpm/min is centrifuged 6min, and liquid is gone to stay object, and the dry 3min of air at room temperature is dissolved in 25 μ L DEPC water, is placed in -80 DEG C
It saves.
Embodiment 2
A method of extracting anopheles total serum IgE, comprising the following steps:
(1) the DEPC aqueous solution of 10%SDS is prepared, for use；
(2) preparation 2moL/L, DEPC aqueous solution, that is, Buffer B solution of pH=3.8 sodium acetate, for use；
(3) it prepares Buffer solution A: sequentially adding 200 μ L DEPC into No.1 1.5mL RNase-Free centrifuge tube
Water, 100 μ L10%SDS, 500 μ L water-saturated phenols, 5 μ L beta -mercaptoethanols, concussion is uniformly mixed up and down, and it is molten to obtain Buffer A
Liquid is placed on ice chest stand-by；
(4) it prepares Buffer C solution: sequentially adding water-saturated phenol, chlorine into No. two 1.5mL RNase-Free centrifuge tubes
Imitative and isoamyl alcohol, concussion is uniformly mixed up and down, obtains Buffer C solution, for use；Wherein, water-saturated phenol, chloroform and isoamyl alcohol
Volume ratio be 125:24:1；
(5) coarse extraction of anopheles total serum IgE: taking 10 hungry work anopheles for raising three days, be put into new RNase-Free from
Heart pipe is placed in -20 DEG C of refrigerator 5min, and then careful take out is placed in No.1 1.5mL RNase-Free centrifuge tube, and grinding is abundant,
After being placed at room temperature for 2min, Buffer B solution is added thereto, piping and druming mixes, and is incubated at room temperature 3min, at 4 DEG C, 12000rpm/
Min is centrifuged 15min, supernatant liquor 1；
(6) supernatant liquor 1 after step (5) centrifugation is transferred to completely in No. three 1.5mL RNase-Free centrifuge tubes,
Isometric Buffer C solution is added thereto, piping and druming mixes, vibrates 15s after covering tightly, be stored at room temperature 5min；At 4 DEG C,
12000rpm/min is centrifuged 15min, obtains supernatant liquor 2, the supernatant liquor 2 after centrifugation is transferred to No. four 1.5mL completely
In RNase-Free centrifuge tube, isometric chloroform is added and mixes well, is stored at room temperature 3min, at 4 DEG C, 12000rpm/min,
It is centrifuged 10min, obtains supernatant liquor 3, the supernatant liquor 3 after centrifugation is transferred to No. five 1.5mL RNase-Free centrifugations completely
Isometric butyl glycol ether is added in Guan Zhong, and the centrifuge tube that turns upside down after covering tightly extremely mixes, -20 DEG C of standing 30min, at 4 DEG C,
12000rpm/min is centrifuged 10min, liquid is gone to stay sediment, is precipitated with the ethanol washing that volumetric concentration is 80%, at 4 DEG C,
7500rpm/min is centrifuged 6min, and liquid is gone to stay object, and the dry 3min of air at room temperature is dissolved in 35 μ L DEPC water, is placed in -80 DEG C
It saves.
Detect example 1
Measure method and tradition SDS method, TRIzoL method, kit of the invention respectively using ultraviolet specrophotometer
(TIANGEN) concentration, OD260/OD280, OD260/OD230 value of the total serum IgE sample extracted；And utilize agarose gel electrophoresis
Detect its integrality；It selects anopheles β-actin gene to be expanded, further verifies the mentioned total serum IgE of the method for the present invention and whether can
Enough meet the requirement of the molecular biology experiments such as RT-PCR.
Using ultramicron ultraviolet-uisible spectrophotometer measure respectively according to 1 step of embodiment extract anopheles total serum IgE with
The quality for the RNA that traditional SDS method, TRIzoL method and (TIANGEN) DNA/RNA IsoLation Kit (centrifugal column type) are extracted,
Result is indicated with the concentration of total serum IgE, OD260/OD280, OD260/OD230 value.
As the result is shown: measured through Nandrop2000, method of the invention and tradition SDS method, TRIzoL method and
(TIANGEN) DNA/RNA IsoLation Kit (centrifugal column type) method is compared, and method of the invention is extracted as known from Table 1
The OD260/OD280 value of total serum IgE is 1.87, and within the scope of ideal value 1.8-2.0, this illustrates the RNA that method of the invention is extracted
Quality is higher, and the substances such as protein, phenol residual is less, seldom degrades；OD260/OD230 value is 2.06 > 2, illustrates this hair
Bright method is extracted in RNA without salt or organic solvent pollution.The OD260/OD280 value that traditional SDS method extracts RNA is 2.11 >
There are RNA signs of degradation for 2 explanations, and OD260/OD230 value is 1.81 < 2.Traditional TRIzoL method extracts the OD260/ of RNA
OD280 value is 1.96, but OD260/OD230 value is 0.41 < 2.The OD260/ for the anopheles RNA that kit (TIANGEN) extracts
OD280 value is that 2.05, OD260/OD230 value is 0.94 < 2.Therefore, traditional SDS method, TRIzoL method and RNA isolation kit
(TIANGEN) it is subsequent be required to carry out the secondary extracting of phenol chloroform and etc. purification of nucleic acid can just carry out subsequent test, and this
The RNA that the method for invention is extracted is with high purity, integrality is good, can directly carry out follow-up test.It the results are shown in Table 1；
Table 1: method of the invention and tradition SDS method, tradition TRIzoL method, RNA isolation kit (TIANGEN) extraction anopheles are total
RNA
Concentration and comparison or purity
| Method | Concentration (ug/uL) | OD260/OD280 | OD260/OD230 |
| Improved method | 1.1980±0.3521 | 1.8733±0.2043 | 2.0616±0.2676 |
| Traditional SDS method | 0.9317±0.2650 | 2.1110±0.1579 | 1.8150±0.2676 |
| Traditional TRIzoL method | 0.6151±0.1862 | 1.9683±0.1600 | 0.4050±0.0367 |
| RNA isolation kit | 0.3005±0.8045 | 2.0550±0.2045 | 0.9400±0.2340 |
Note: every group of data are the average value of 40 repetition experiments, and " ± " number subsequent numerical value represents standard error, every line number
Value examines difference (P=0.05) by one-way analysis of variance.
Detect example 2
The anopheles total serum IgE and tradition SDS method, TRIzoL method and (TIANGEN) DNA/RNA that 1 step of embodiment is extracted
The anopheles total serum IgE that IsoLation Kit (centrifugal column type) is extracted carries out agarose gel electrophoresis detection.Method: 1.5% fine jade of preparation
Sepharose takes the RNA sample of 5 μ L and 6 × Loading buffer of 1 μ L to be mixed, 110V, 300mA electrophoresis 20min,
Gel imager is imaged and takes pictures.
Its agarose gel electrophoresis testing result is shown in Fig. 2, as shown in Fig. 2, swimming lane D is that the anopheles that the method for the present invention is extracted is total
RNA integrality is preferable, and electrophoresis result shows that 28S rRNA, 18S rRNA and 5S rRNA band are completely clear, without bright between band
Aobvious diffusing phenomenon, then illustrate that integrality is good；Swimming lane C is the anopheles total serum IgE that tradition SDS method is extracted, except 18S rRNA and 5S rRNA
Outside two clear bands, 28S rRNA is weaker, and there are apparent DNA pollution, integrality is bad, of low quality.Swimming lane B is examination
The anopheles total serum IgE that agent box method (TIANGEN) is extracted, 18S rRNA and 28S rRNA band is faint, and 5S rRNA is lacked, completely
Property is bad, of low quality.Swimming lane A is the anopheles total serum IgE that tradition TRIzoL method is extracted, only visible 18S rRNA and 5S rRNA two
Band shows that integrality is bad.Swimming lane M is D2000Marker.
Detect example 3
β-actin gene primer the sequence (being shown in Table 2) that the extracted total serum IgE of embodiment 1 is amplified after RT-PCR, inspection
Survey whether band is single, neat and whether there is or not tractions.
Method: β-actin gene primer sequence is used, by the extracted total serum IgE sample of embodiment 1 and tradition TRIzoL method
The total serum IgE sample of direct extraction synthesizes cDNA by RT-PCR kit specification, then carries out PCR by template of cDNA.PCR is anti-
The system is answered to include: 1.0 μ L cDNA templates, 0.5 μ L upstream primer, 0.5 μ L downstream primer, 6.25 μ 2 × Taq of L Master
Mix, 4.25 μ L are without enzyme water.PCR reaction condition: 94 DEG C of initial denaturation 3min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C extend
30s is recycled 35 times；72 DEG C of terminals extend 7min；4 DEG C of preservations.Amplified production carries out 1.5% agarose gel electrophoresis analysis, purple
Result is analyzed under outer light.({ Hu Ying, Shen Bo, Yang Mingxia wait Culex pipiens pallens β-flesh to β-actin gene primer sequence reference literature
Filamentous actin gene cloning and sequencing [J] China preventing and treating verminosis magazine, 2003,16 (19): 65-68. }).
2 β-actin gene primer sequence of table
| Primer | Sequence (5 ' -3 ') |
| Upstream primer | ATGGGCCAGAAGGACTCGTA |
| Downstream primer | GCAGATTCCATACCCAAGAAGG |
The result shows that: RT-PCR testing result is shown in Fig. 3, as shown in figure 3, swimming lane A, B, C, D are what the method for the present invention was extracted
Product after the amplification of anopheles total serum IgE, swimming lane M1 are DL2000Marker；Thus the anopheles that can be shown that method of the invention is extracted is total
β-actin genetic fragment the length that RNA is amplified after RT-PCR is 240bp or so, consistent with expected results, and band list
One, neat, without traction, it can be used for the later experiments such as cDNA library building, Northern hybridization analysis.
The present invention is exemplarily described above, it is clear that present invention specific implementation is not subject to the restrictions described above,
As long as using the improvement for the various unsubstantialities that the inventive concept and technical scheme of the present invention carry out, or not improved this is sent out
Bright conception and technical scheme directly apply to other occasions, within the scope of the present invention.Protection of the invention
Range should be determined by the scope of protection defined in the claims.
Claims (10)
1. a kind of method for extracting anopheles total serum IgE, which is characterized in that specific step is as follows:
1) the hungry anopheles raised is taken, then freezing processing the anopheles after freezing is added in Buffer solution A, piping and druming is mixed
It is even, it is stored at room temperature, obtains anopheles tissue homogenate；
2) Buffer B solution is first added into obtained anopheles tissue homogenate, is successively mixed through piping and druming, is incubated at room temperature, centrifugation
1, supernatant liquor 1 is extracted into centrifuge tube, then Buffer C solution is added into supernatant liquor 1, successively mix through piping and druming, room temperature
It is incubated for, centrifugation 2, supernatant liquor 2 is extracted into centrifuge tube, then Buffer solution D is added into supernatant liquor 2, successively through blowing and beating
It mixes, incubation at room temperature, centrifugation 3, extracts supernatant liquor 3 into centrifuge tube, then Buffer E solution is added into supernatant liquor 3,
Successively being mixed through piping and druming, refrigeration is incubated for, and centrifugation 4 goes liquid to stay sediment, Buffer F solution is added in last sediment upwards,
It is successively mixed through piping and druming, centrifugation 5 goes liquid to stay object, drying at room temperature 3-5min is dissolved in 20-30 μ L DEPC water in subzero -75-
It is saved at 85 DEG C.
2. the method according to claim 1 for extracting anopheles total serum IgE, which is characterized in that the freezing processing: temperature zero
It is 15-25 DEG C lower, duration 3-10min.
3. the method according to claim 1 for extracting anopheles total serum IgE, which is characterized in that the Buffer solution A is by 100
The lauryl sodium sulfate (SDS) of μ L concentration 10%, 500 μ L water-saturated phenols, 200 μ L EPC water and 5 μ L beta -mercaptoethanols are constituted.
4. the method according to claim 1 for extracting anopheles total serum IgE, which is characterized in that the Buffer B solution is vinegar
Sour sodium buffer, concentration 2moL/L, pH 3.2-3.9.
5. the method according to claim 1 for extracting anopheles total serum IgE, which is characterized in that the Buffer C solution includes
Following component: water-saturated phenol, chloroform, isoamyl alcohol, and volume ratio is 125:24:1.
6. the method according to claim 1 for extracting anopheles total serum IgE, which is characterized in that the Buffer solution D is chlorine
Imitative, Buffer E solution is butyl glycol ether, and Buffer F solution is ethyl alcohol.
7. it is according to claim 1 extract anopheles total serum IgE method, which is characterized in that it is described piping and druming mix when it is a length of
12-18s。
8. it is according to claim 1 extract anopheles total serum IgE method, which is characterized in that it is described centrifugation 1-4 revolving speed be
12000rpm/min, temperature are 4 DEG C, time 10-15min, and the revolving speed for being centrifuged 5 is 7500rpm/min, and temperature is 4 DEG C, the time
For 5-7min.
9. the method according to claim 1 for extracting anopheles total serum IgE, which is characterized in that the duration 3- of the incubation at room temperature
5min, the temperature for refrigerating incubation is subzero 18-23 DEG C, duration 25-35min.
10. the method according to claim 1 for extracting anopheles total serum IgE, which is characterized in that the quantity of the anopheles is 3-20
Only.
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