CN108251414A - The extracting method and extracts kit of bone marrow cell genomic DNA - Google Patents
The extracting method and extracts kit of bone marrow cell genomic DNA Download PDFInfo
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Abstract
The present invention provides a kind of extracting method and extracts kit of bone marrow cell genomic DNA, extracting method includes：Using Proteinase K and lysate processing bone marrow cell sample, then rnase digestion is used, it is put into silica gel adsorption column and adsorbs after being mixed with absolute ethyl alcohol, handled successively using the first cleaning solution, the second cleaning solution and eluent, obtain bone marrow cell genomic DNA after purification.The extracting method of the bone marrow cell genomic DNA of the present invention can extract the gDNA that quality is high, purity is high, extraction effect is reliable and stable by controlling formula, adjusting pH value and ion concentration etc. in the case of low cost.
Description
Technical field
The invention belongs to DNA to extract field, is related to a kind of extracting method and extracts reagent of bone marrow cell genomic DNA
Box.
Background technology
Bone marrow cell is one of cell important in human body, understand bone marrow cell genomic DNA (Genomic DNA,
GDNA) contribute to the health condition for understanding human body, therefore, bone marrow cell gDNA is first extracted with regard to necessary.
People often extract bone marrow cell gDNA with bone marrow cell gDNA extracts kits.The marrow of commercial type at present
Or cell gDNA extracts kits are sacrificed its purity in order to improve the concentration of gDNA or are sacrificed to improve its purity
Its concentration.The concentration and purity of gDNA can build library and sequencing data has an impact to subsequent, therefore, same when extracting gDNA
When obtain higher concentration and larger purity gDNA it is just very significant.
Invention content
The defects of it is an object of the invention to overcome the prior art, provides a kind of extraction of bone marrow cell genomic DNA
Method can obtain higher concentration and the gDNA of larger purity simultaneously in the case of low cost.
Second object of the present invention is to provide a kind of bone marrow cell extracting genome DNA that can realize the above method
Kit.
To achieve the above object, the present invention uses following technical scheme：
A kind of extracting method of bone marrow cell genomic DNA, includes the following steps：
(1), bone marrow cell sample, Proteinase K Solution and lysate are mixed, cracking suspension is obtained after cracking；
(2), cracking suspension is cooled to room temperature, after mixing and digest with ribalgilase, obtains digestive juice；
(3), digestive juice with absolute ethyl alcohol is mixed, adds in silica gel adsorption column and carry out as sample solution after solution clarification
Absorption cleans silica gel adsorption column using the first cleaning solution and the second cleaning solution successively and discards waste liquid；
(4), the silica gel adsorption column obtained using elution step (3), isolated bone marrow cell genomic DNA.
Wherein, in above-mentioned extracting method, the volume ratio of bone marrow cell sample and Proteinase K Solution can be (5-
20):1, can be (8-15):1, or 10:1.The concentration of Proteinase K can be 20 ± 10mg/ in Proteinase K Solution
Ml can also be 20 ± 5mg/ml, or 20mg/ml.The volume ratio of bone marrow cell sample and lysate can be 1:
(0.5-2) can be 1:1.
The solvent of above-mentioned lysate is distilled water, and solute includes：Guanidine hydrochloride, NaTDC, ionic surfactant
Agent and nonionic surface active agent.The concentration of guanidine hydrochloride in lysate can be 3-10M, can be 3.5-9.5M, may be used also
Think 5-9M, can be further 5.5-7.5M.The quality percent by volume of NaTDC can be 0.1- in lysate
0.5%, it can be 0.2-0.4%, more can be 0.25-0.3%.Ionic surfactant is sodium N-lauroyl sarcosinate,
Quality percent by volume in lysate is 0.8%-1.2%.Nonionic surface active agent is polyoxyethylene ether, is being cracked
Quality concentration of volume percent in liquid is 0.4%-0.5%.
Mixed liquor of first cleaning solution for guanidine hydrochloride and absolute ethyl alcohol, pH value 9.0-10.0.Optionally, the first cleaning solution
The molar concentration of middle guanidine hydrochloride is more than the molar concentration of guanidine hydrochloride in lysate, and optionally, guanidine hydrochloride is dense in the first cleaning solution
Degree is the concentration 1.4 of guanidine hydrochloride in lysate again to 2 times.The volume ratio of guanidine hydrochloride and absolute ethyl alcohol is 1 in first cleaning solution:
(0.9‐1)。
Mixed liquor of second cleaning solution for Tris-HCl buffer solutions and absolute ethyl alcohol, pH value 7.5-8.5.Tris-HCl delays
The concentration of trishydroxymethylaminomethane (Tris) can be 100 ± 10mM in fliud flushing.Tris-HCl buffer solutions and absolute ethyl alcohol
Volume ratio is 1:(3-7), or 1:(4‐5).
Eluent is trishydroxymethylaminomethane (Tris) and the aqueous solution of ethylenediamine tetra-acetic acid (EDTA).Optionally, it washes
A concentration of 10 ± 1mM of trishydroxymethylaminomethane (Tris) in de- liquid.A concentration of 0.8-1mM of EDTA in eluent.
A concentration of working concentration of above-mentioned various reagents.
A kind of bone marrow cell genome DNA extracting reagent kit, including following reagent：Proteinase K reagents, lysate, core
Ribonuclease T. reagent, the first cleaning solution, the second cleaning solution, eluent and silica gel adsorption column.
Wherein, protease reagent contains the Proteinase K of 20 ± 10mg/mL.
The solvent of lysate be distilled water, solute include guanidine hydrochloride, NaTDC, ionic surfactant and it is non-from
Subtype surfactant.The concentration of guanidine hydrochloride in lysate can be 3-10M, or 3.5-9.5M can also be 5-
9M can be further 5.5-7.5M.The quality percent by volume of NaTDC can be 0.1-0.5% in lysate,
It can be 0.2-0.4%, more can be 0.25-0.3%.Ionic surfactant is sodium N-lauroyl sarcosinate, is being split
The quality percent by volume solved in liquid can be 0.8%-1.2%.Nonionic surface active agent is polyoxyethylene ether, is being split
The quality percent by volume solved in liquid can be 0.4%-0.5%.
Mixed liquor of first cleaning solution for guanidine hydrochloride and absolute ethyl alcohol, pH value 9.0-10.0.Hydrochloric acid in first cleaning solution
The concentration of guanidine is more than the concentration of guanidine hydrochloride in lysate.
Mixed liquor of second cleaning solution for Tris-HCl buffer solutions and absolute ethyl alcohol, pH value 7.5-8.5.Tris-HCl delays
A concentration of 100 ± 10mM of trishydroxymethylaminomethane (Tris) in fliud flushing.
Eluent is trishydroxymethylaminomethane (Tris) and the aqueous solution of ethylenediamine tetra-acetic acid (EDTA).Three in eluent
A concentration of 0.8-1mM of a concentration of 10 ± 1mM of hydroxymethyl aminomethane (Tris), EDTA.
The invention has the advantages that：
Firstth, the present invention makes protein denaturation using the GuHCl of high concentration, and destruction membrane structure is unlocked simultaneously to be connected with nucleic acid
The protein connect is free in so as to fulfill nucleic acid in cracking liquid system, and using protease by protein digestibility into small segment,
Promote separating for nucleic acid and protein.In order to obtain purer nucleic acid, detergent, anion surfactant and nonionic are selected
Surfactant is used in combination, and further lytic cell, soluble protein, the albumen that some can particularly be made to be insoluble in water
Dissolving.
Secondth, the present invention is using Silicon moulds column method purification of nucleic acid, under high PH washing lotions (the first cleaning solution) with high salt, nucleic acid
Specificity is attached on pellosil, and broken cell fragment and albumen can not be but attached on film, isolated with this nucleic acid
Come.Under the low PH washing lotions of less salt (the second cleaning solution), impurity and remaining salt that previous step does not wash away can be further removed
Ion.
Third, the eluent present invention employs optimization, under the incubation of the eluent, nucleic acid is discharged into from pellosil
In eluent, the genomic DNA of high-purity high-concentration is obtained under high speed centrifugation
In short, the extracting method of the bone marrow cell genomic DNA of the present invention is dense by controlling formula, adjusting pH value and ion
Degree etc. can extract the gDNA that quality is high, purity is high in the case of low cost, and extraction effect is reliable and stable.
Description of the drawings
Fig. 1 is lifted the agarose gel electrophoresis comparison diagram of DNA by the embodiment of the present invention 1 and the prior art.
Specific embodiment
The present invention provides a kind of extracting method and extracts kit of bone marrow cell genomic DNA, for thin to marrow
Born of the same parents' genomic DNA extracts, and obtains the bone marrow cell genomic DNA (gDNA) with high concentration and high-purity.
<The extracting method of bone marrow cell genomic DNA>
A kind of extracting method of bone marrow cell genomic DNA, includes the following steps：
(1), cracking suspension is obtained after bone marrow cell sample, Proteinase K and lysate being mixed and cracked；
(2), cracking suspension is cooled to room temperature, after mixing and digest with ribalgilase, obtains digestive juice；
(3), digestive juice with absolute ethyl alcohol is mixed, adds in silica gel adsorption column and carry out as sample solution after solution clarification
Absorption；
(4), the silica gel adsorption column that the first cleaning solution and the second cleaning solution cleaning step (3) obtain is used successively and is discarded useless
Liquid；
(5), the silica gel adsorption column obtained using elution step (4), isolated bone marrow cell genomic DNA.
Wherein, the purpose of step (1) is cracking bone marrow cell, and nucleic acid therein is made to dissociate into cracking suspension.
In step (1), contain Proteinase K Solution for cracking in the cracking system of bone marrow cell.Proteinase K is a kind of
Protein digestibility can be small for the degradation of protein in biological sample by the highly active protein enzyme of Subtilisin enzyme
Segment promotes separating for nucleic acid and protein.The concentration of Proteinase K can be 20 ± 10mg/ml in Proteinase K Solution, may be used also
Think 20 ± 5mg/ml, or 20mg/ml.At this point, the volume ratio of bone marrow cell sample and Proteinase K Solution can be
(5‐20):1, preferably (8-15):1, more preferably 10:1.For example, when detecting, if the volume of bone marrow cell sample is 200 μ
L, then the concentration of Proteinase K Solution can be 20mg/ml, and volume can be 20 μ l.
In step (1), also contain lysate in cracking system.The volume ratio of bone marrow cell sample and lysate can be
1:(0.5-2) can also be 1:1.For example, if the volume of bone marrow cell sample is 200 μ l, the volume of lysate can be
200μl.Lysate is a kind of composite solution combined by many kinds of substance, and solute includes：Guanidine hydrochloride, NaTDC, ion
Type surfactant and nonionic surface active agent, solvent are distilled water.After all solutes are dissolved in distilled water, 0.22 need to be used
μm aqueous membrane filtration after can be used as lysate.
Wherein, the concentration of the guanidine hydrochloride in lysate (Guanidine hydrochloride, GuHcl) is higher, Ke Yiwei
3-10M, or 3.5-9.5M can also be 5-9M, can also be further 5.5-7.5M.The guanidine hydrochloride of high concentration plays
Make the effect of the protein denaturation in cracking system, the protein being connected with nucleic acid also unlocked while membrane structure is destroyed,
It is free in cracking suspension so as to fulfill nucleic acid.
Ionic surfactant is sodium N-lauroyl sarcosinate (Sodium lauroyl sarcosine), belongs to cloudy
Ionic surfactant.In some embodiments, the quality percent by volume in lysate generally can be 0.8%-
1.2%, but in further embodiments, working concentration when cracking may be 1.6mg/ μ l-2.4mg/ μ l.Matter herein
Amount concentration of volume percent refers to the ionic surfactant containing 0.8-1.2mg in the lysate of 100ml.
Nonionic surface active agent is polyoxyethylene ether (Brij-58), in some embodiments, in lysate
Quality percent by volume generally can be 0.8%-1.2%, but in further embodiments, working concentration when cracking also may be used
Think 0.8mg/ μ l-1.0mg/ μ l.Quality concentration of volume percent herein refers to containing 0.8- in the lysate of 100ml
The nonionic surface active agent of 1.2mg.Below similarly.
Ionic surfactant and nonionic surface active agent can make the protein and NaTDC after denaturation
It is dissolved in lysate with reference to after.
In some embodiments, matter of the NaTDC (Deoxycholate) as ionic detergent in lysate
It can be 0.1-0.5% to measure percent by volume, can also be 0.2-0.4%, or 0.25-0.3%.Suitable concentration takes off
Oxycholic acid sodium can either ensure that the removal of denatured protein, and can prevent from adding in excessive ionic and non-ionic surface
Activating agent, so as to prevent these surfactants from being had adverse effect on to subsequent purification process.
Experiment proves：Ionic, nonionic surface active agent and being used in combination for detergent can further crack carefully
Born of the same parents and soluble protein can particularly dissolve some albumen for being insoluble in water.
Lysate can provisional configuration before actual use, as long as the working concentration of various components meets above-mentioned condition i.e.
It can.
In step (1), bone marrow cell sample, Proteinase K and lysate are sufficiently mixed, obtain mixed liquor, Zhi Houxu
Cracking process is carried out, specially：By above-mentioned mixed liquor under conditions of 65 DEG C water-bath about 15 minutes.Recommend every 5 minutes
After take out reverse concussion mixing, and of short duration wink from rear continuation water-bath, treat to obtain cracking suspension after water-bath.Of short duration wink from
Condition be：Centrifugal rotational speed is less than 4000rpm, and centrifugation time is less than or equal to 5s.Of short duration wink sticks from when can make and overturn mixing
Mixed liquor on lid is come back in pipe.Low speed and short time can ensure that the substance in mixed liquor is unlikely to because of centrifugation
The effect of power and deposit to tube bottom, effect be with can making sample dissociation more fully.
In step (1), due to the sticky of bone marrow cell sample and the special circumstances presence such as improper is preserved, if pyrolysis time
Too short, the cracking that may cause sample is insufficient, and nucleic acid yield is low；If pyrolysis time is long, the drop of nucleic acid molecules may be caused
Solution, the uncomfortable situation of fracture.Therefore, it is recommended that pyrolysis time be 15 to 20 minutes.It must ensure bone marrow cell before water-bath cracking
The abundant mixing of sample and lysate observes the variation of bone marrow cell sample color in cracking during cracking.If marrow is thin
Born of the same parents' sample is no before relatively cracking to occur apparent color change, pyrolysis time can be appropriately extended 5 minutes.For special sample (bone
Marrow preserve improper, quality it is bad when sample), give the pyrolysis time of most 20 minutes, otherwise DNA is very easy to drop
Solution, so as to influence the accuracy of result.
In step (2), concussion mixing is overturned after the cracking suspension taking-up that step (1) is obtained, of short duration wink is from room
Temperature place 2-3 minute, treat that the cracking suspension is cooled to room temperature, i.e., addition Ribonuclease in Aqueous Solution (Ribonuclease,
RNase).The concentration of Ribonuclease in Aqueous Solution can be 100-500mg/ml, can also be 200-300mg/ml.Ribalgilase
Additive amount depending on the additive amount of bone marrow cell.The volume of ribalgilase and the volume ratio of bone marrow cell sample can be
1:(50-250), or 1:(100‐200).For example, when the volume of bone marrow cell sample is 200 μ l, ribalgilase
Volume be 2 μ l.Ribalgilase interferes DNA to avoid it for the RNA present in cracking suspension that degrades.
In step (2), the digestion process of ribalgilase includes the following steps：It will be cooled to the cracking suspension of room temperature
After being mixed with Ribonuclease in Aqueous Solution, concussion mixing is overturned, of short duration wink digests 5 to 10 minutes to get to digestive juice from, room temperature.
In step (3), the volume of absolute ethyl alcohol is more than the volume of bone marrow cell sample, can be bone marrow cell sample
The 110-120% of volume.For example, if the volume of bone marrow cell sample is 200 μ l, the volume of absolute ethyl alcohol can be 220 μ l.
After absolute ethyl alcohol is added in, fully reverse mixing, until after existing without precipitation or apparent floccule, of short duration wink from.Of short duration wink from
Purpose be provided to make the liquid on lid to return in pipe, be both lysate sample is made full use of and is prevented experiment sample
The pollution and cross contamination of product.Precipitation will not be generated in the step but is not excluded for that apparent transparent floccule may be generated, at this time
Fully shaking mixing must be obtained until transparent floccule disappears, then obtained mixed liquor is added in ethanol on pellosil post.Absolute ethyl alcohol is not
Nucleic acid can be dissolved, for dissolving and removing broken cell impurities, albumen etc..
In step (3), after absolute ethyl alcohol is added in digestive juice, floccule can significantly occur, should overturn at this time
Mixing is blown and beaten with pipettor repeatedly until occurring without apparent floccule, this step carries out at room temperature, it is not necessary to be incubated at 4 DEG C
It educates.The prior art uses the precipitation method, with ethyl alcohol/isopropanol precipitating nucleic acid, for obtain nucleic acid need ethyl alcohol/isopropanol is same
Sample cracks mixed liquor and is incubated 10-30 minutes in 4 DEG C, this step allows for that carried sample nucleic acid amount is low and the step that need to carry out
Suddenly.The present invention considers the particularity of sample of bone marrow, better quality, and it is few (such as that sample size is extracted under laboratory condition：At least
200 μ l are extracted), yield meets requirement of experiment, therefore is incubated at room temperature, than prior art more convenient and quicker.
The main purpose of step (4) is to wash the unadsorbed denatured protein on silica gel adsorption column and other impurity.
Cleaning process is divided into two steps, and the first step is washed using the first cleaning solution, and second step is washed using the second cleaning solution.
Wherein, mixed liquor of first cleaning solution for guanidine hydrochloride and absolute ethyl alcohol, pH value 9.0-10.0, for example, 9.0 ±
0.1, the volume ratio of guanidine hydrochloride and absolute ethyl alcohol is 1:(0.9‐1).The molar concentration of guanidine hydrochloride is more than cracking in first cleaning solution
The molar concentration of guanidine hydrochloride in liquid is preferably the concentration 1.4 of guanidine hydrochloride in lysate again to 2 times.If for example, in lysate
Guanidine hydrochloride a concentration of 3.5M, then the concentration of guanidine hydrochloride can be 5M in the first cleaning solution.First cleaning solution is high pH with high salt
It is worth washing lotion, its purpose is to remove denatured protein and impurity, because under conditions of with high salt and high ph-values, nucleic acid molecules energy
It is enough specifically binding on silica gel absorption film, and broken cell fragment and protein can not be attached on film, it therefore, can
Nucleic acid specificity to separate.In order to ensure not wash accidentally lower nucleic acid, nucleic acid yield is improved, therefore uses absolute ethyl alcohol and salt
Sour guanidine mixes.It can repeatedly be added in the first cleaning solution as the case may be and be washed, and sample solution can be made in practice
Repeatedly pass through silica gel absorption film.If the volume of bone marrow cell sample is 200 μ l, the addition volume of the first cleaning solution can be 500 μ
l。
Mixed liquor of second cleaning solution for Tris-HCl buffer solutions and absolute ethyl alcohol, pH value 7.5-8.5, for example, 7.5
±0.1.A concentration of 100 ± 10mM of trishydroxymethylaminomethane (Tris) in Tris-HCl buffer solutions.Tris-HCl buffer solutions
Volume ratio with absolute ethyl alcohol is 1:(3-7), preferably 1:(4‐5).Second cleaning solution is less salt low ph value washing lotion, the purpose is to
For the impurity for washing away guanidine hydrochloride and not washing away so that the absorption UF membrane of guanidine hydrochloride and impurity and silica gel adsorption column is matched simultaneously
Adjustment pH value is closed, nucleic acid is enabled to not washed lower adsorbed film accidentally, improves the yield of nucleic acid.The preservation pH that DNA can dissolve is 9.0
Left and right adjusts the pH value of the second cleaning solution between 7.5-8.5, particularly 7.5 ± 0.1, and guarantee is not washed lower combination accidentally and inhaled
DNA on membrane.It can also repeatedly add the second cleaning solution according to actual conditions and washed.If the body of bone marrow cell sample
Product is 200 μ l, then the addition volume of the second cleaning solution can be 500 μ l.
After the cleaning process by (4) step, nucleic acid molecules are specifically adsorbed on adsorbed film, and denatured protein
It is all washed off with impurity, therefore, the purpose of (5) step is that the nucleic acid molecules of absorption are eluted and are discharged into using eluent
In eluent.Aqueous solution of the eluent for trishydroxymethylaminomethane (Tris) and ethylenediamine tetra-acetic acid (EDTA), in eluent
A concentration of 10 ± 1mM of trishydroxymethylaminomethane (Tris), a concentration of 0.8-1mM of EDTA in eluent.Trihydroxy methyl ammonia
The purpose of methylmethane (Tris) is that nucleic acid molecules is made to be separated from adsorbed film, and a stable preservation is provided for nucleic acid molecules
Environment.The concentration of ethylenediamine tetra-acetic acid (EDTA) is relatively low, can inhibit the activity of nuclease so that nucleic acid molecules are not degraded.
Eluent after elution takes precipitation after high speed centrifugation, which is the bone marrow cell gene with high-purity and high concentration
Group DNA.According to the volume of bone marrow cell sample, the addition volume of eluent can be the 1/4 to 1/ of bone marrow cell sample volume
2.For example, the volume of bone marrow cell sample is 200 μ l, then the volume of eluent can be 50-100 μ l.
<Bone marrow cell genome DNA extracting reagent kit>
A kind of bone marrow cell genome DNA extracting reagent kit, it is suitable for the extractions of above-mentioned bone marrow cell genomic DNA
Method, including following reagent：Proteinase K reagents, lysate, ribonucleic acid enzymatic reagent, the first cleaning solution, the second cleaning solution,
Eluent, silica gel adsorption column and waste collection pipe.Silica gel adsorption column is removably nested in waste collection pipe.Wherein, albumen
Enzyme K reagents contain Proteinase K.The concentration of Proteinase K can be 20 ± 10mg/mL, preferably 20 ± 5mg/ in proteinase K reagents
ML, more preferably 20mg/mL.In actual use, the additive amount of proteinase K reagents is：Bone marrow cell sample is tried with Proteinase K
The volume ratio of agent can be (5-20):1, preferably (8-15):1, more preferably 10:1.
Lysate makes the substances such as nucleic acid, protein be escaped from cell, including hydrochloric acid for cracking bone marrow cell sample
Guanidine, NaTDC, ionic surfactant and nonionic surface active agent.A concentration of 3- of guanidine hydrochloride in lysate
10M, preferably 3.5-9.5M, more preferably 5-9M, further preferably 5.5-7.5M.The quality of NaTDC in lysate
Percent by volume is 0.1-0.5%, preferably 0.2-0.4%, more preferably 0.25-0.3%.Ionic surfactant is the moon
Osmanthus sarcosinate, the concentration in lysate can be 0.8%-1.2%, can also be 0.9%-1.1%, can also
It is 1.0%.Nonionic surface active agent is polyoxyethylene ether, a concentration of 0.4%-0.5% in lysate, can also
It is 0.45%.
First cleaning solution is guanidine hydrochloride and the mixed liquor of absolute ethyl alcohol, and pH value can be 9.0 ± 0.1.In first cleaning solution
The concentration of guanidine hydrochloride is more than the concentration of guanidine hydrochloride in lysate.
Second cleaning solution is Tris-HCl buffer solutions and the mixed liquor of absolute ethyl alcohol, and pH value can be 7.5 ± 0.1.Tris‐
A concentration of 100 ± 10mM of trishydroxymethylaminomethane (Tris) in HCl buffer solutions.
Eluent is trishydroxymethylaminomethane (Tris) and the aqueous solution of ethylenediamine tetra-acetic acid (EDTA).Three in eluent
A concentration of 10 ± 1mM of hydroxymethyl aminomethane (Tris).A concentration of 0.8-1mM of EDTA in eluent.
The concentration of above-mentioned each reagent may each be working concentration, not store concentration.For storage concentration, there is no assorted
Requirement, as long as being made into working concentration at work.
The ingredient of bone marrow cell genome DNA extracting reagent kit is as shown in table 1 below：
The component list of 1 bone marrow cell genome DNA extracting reagent kit of table
In upper table, phonetic abbreviations of the Y for cloud health Gene Technology (Shanghai) Company Limited, initials of the L for lysis, W
The first cleaning solution is represented for the English initial of WASH washing lotions, 1, and 2 represent the second cleaning solution, and E represents the English of elution eluents
Literary initial.
Lysate needs to have seen whether precipitation before use, if any precipitation, in 37 DEG C of water-baths until precipitation disappears, rocks
It is used after even.First cleaning solution and the second cleaning solution can Extemporaneous before the use.
Mentioned reagent can not only prepare in advance sells, but also can Extemporaneous when in use as finished product.It is as long as various
The concentration of substance meets above-mentioned requirements.
<Embodiment 1>
It further illustrates the present invention with reference to embodiments.
A kind of extracting method of bone marrow cell genomic DNA is present embodiments provided, is included the following steps：
(1), 200 μ l bone marrow cell samples are added in into a clean 1.5ml centrifuge tubes, it is dense then to sequentially add 20u l
The Proteinase K Solution and 200 μ l lysates for 20mg/ml are spent, fully reverse concussion mixing, of short duration wink is from acquisition mixed liquor；
(2), the mixed liquor that step (1) obtains was taken out in 65 DEG C of water-baths 15 minutes during heating water bath every 5 minutes
Reverse concussion mixing, of short duration wink crack from rear continuation water-bath, treat to obtain cracking suspension after water-bath；
(3) concussion mixing is overturned after the cracking suspension obtained by step (2) is taken out, of short duration wink is from being placed at room temperature for 2-3 point
Clock after being cooled to room temperature, adds in the RNase A of 2 a concentration of 200mg/ml of μ l, overturns concussion mixing, of short duration wink is from room temperature disappears
Change 5 minutes, obtain digestive juice；
(4), the absolute ethyl alcohol of 220 μ l is added in into centrifuge tube, and the fully reverse mixing of digestive juice, until without precipitation or bright
After aobvious floccule exists, of short duration wink is from using gained liquid as in sample solution all addition silica gel adsorption columns, putting on waste collection
Pipe, is placed at room temperature for 1-2 minutes, is centrifuged 90 seconds in 7000 × g (9000rpm) room temperature, discards the waste liquid in waste collection pipe；
(5), silica gel adsorption column is put back in waste collection pipe, 500 the first cleaning solutions of μ l is added in into silica gel adsorption column,
7000 × g (9000rpm) room temperature centrifuges 60 seconds, outwells the waste liquid in waste collection pipe；
(6), silica gel adsorption column is put back in waste collection pipe, 500 the second cleaning solutions of μ l is added in into silica gel adsorption column,
7000 × g (9000rpm) room temperature centrifuges 60 seconds, outwells the waste liquid in waste collection pipe；
(7), silica gel adsorption column is put back in waste collection pipe, 13500 × g (12000rpm) room temperature centrifuges 120 seconds, discards
Silica gel adsorption column is put into clean new 1.5ml centrifuge tubes by waste collection pipe；The silica gel in 1.5ml centrifuge tubes will be sleeved on
Adsorption column is positioned on 25 DEG C of metal heaters, opens the lid of silica gel adsorption column, and dry at least 2 minutes complete to remaining ethyl alcohol
Full volatilization；
(8), to the hanging elution for adding in 50 to 100 μ l and being preheated to 65 DEG C in the silica gel absorption film of silica gel adsorption column center
Liquid is placed at room temperature for 1 minute, is centrifuged 120 seconds in 13500 × g (12000rpm) room temperature, collects precipitation and remembers bone marrow cell gene
Group DNA, in -20 DEG C of preservations.
<Experiment>
It carries out Qubit and agarose gel electrophoresis detection respectively to the bone marrow cell genomic DNA of embodiment 1, obtains real
Test result.Wherein, Qubit and agarose gel electrophoresis detection method are common knowledge, and those skilled in the art can obtain
Specific detection method, so it will not be repeated.
Experiment condition is as follows：The volume of bone marrow cell is 200 μ l, and the volume of eluent is 50 μ l, agarose gel electrophoresis
The condition of detection is：220V, 1% agarose gel electrophoresis, electrophoresis 30min.
Experimental result is as shown in the following table 2 and Fig. 1.
2 experimental result contrast table of table
| Sample ID | Qubit | Unit | Sample Type | Vol(ul) | Total(ug) | 20ng QC | 1.2*Loading |
| Test B | 53.0 | ng/μl | DNA | 32 | 1.70 | 0.38 | 5.0 |
| Test T | 71.4 | ng/μl | DNA | 45 | 3.21 | 0.28 | 5.0 |
In table 2, Vol refers to that 50 μ l eluents add in the volume of the liquid containing DNA obtained after adsorption column centrifugation
(because of loss and error when adsorbed film can remain eluent and final liquid measure, the liquid containing DNA finally obtained
50 μ l eluted when adding in can be less than).20ng QC refer to drawing the DNA+5ul 1.2*Loading Buffer of 20ng, come
With 1% agarose 220v 30min electrophoresis.1.2*Loading Buffer are 6*Loading Buffer (the Cat. ﹟ of TaKaRa:
9156) it is diluted with Nuclease-Free Water.
In table 2, Test B are commercially available tissue extraction agent box, and Test T are the bone marrow cell genome of the present invention
DNA extraction agent boxes.
It is measured by Qubit it is found that the DNA concentration of Test B is 53.0ng/ μ l, and the DNA concentration of Test T is
71.4ng/μl.It follows that the bone marrow cell genome DNA extraction method of the present invention obtains the DNA of high-purity.Same
Different condition (concentration for such as changing lysate, two kinds of cleaning solutions and eluent) is replaced under sample condition and in identical item
It is detected under part and has also obtained same result.
As shown in Figure 1, the band of Test T is significantly narrower than Test B, and band understands, becomes clear, and does not occur miscellaneous band, and DNA compares
Pure, the extraction quality and purity for illustrating Test T are substantially better than Test B.
It is understood that the above description of the embodiments is intended to facilitate those skilled in the art and using this hair
It is bright.Person skilled in the art obviously easily can make various modifications, and described herein to these embodiments
General Principle is applied in other embodiment without having to go through creative labor.Therefore, the present invention is not limited to implementations here
Example, those skilled in the art's announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be
Within protection scope of the present invention.
Claims (10)
1. a kind of extracting method of bone marrow cell genomic DNA, it is characterised in that：It includes the following steps：
(1), bone marrow cell sample, Proteinase K and lysate are mixed, cracking suspension is obtained after cracking；
(2), the cracking suspension is cooled to room temperature, after mixing and digest with ribalgilase, obtains digestive juice；
(3), digestive juice with absolute ethyl alcohol is mixed, adds in silica gel adsorption column and inhaled as sample solution after solution clarification
It is attached, the silica gel adsorption column is cleaned using the first cleaning solution and the second cleaning solution successively and discards waste liquid；
(4), the silica gel adsorption column obtained using elution step (3), isolated bone marrow cell genomic DNA.
2. the extracting method of bone marrow cell genomic DNA according to claim 1, it is characterised in that：The bone marrow cell
The volume ratio of sample and the Proteinase K can be (5-20):1, can be (8-15):1, or 10:1；And/or
The volume ratio of the bone marrow cell sample and the lysate can be 1:(0.5-2) can be 1:1.
3. the extracting method of bone marrow cell genomic DNA according to claim 1, it is characterised in that：The lysate packet
It includes：Guanidine hydrochloride, NaTDC, ionic surfactant and nonionic surface active agent.
4. the extracting method of bone marrow cell genomic DNA according to claim 3, it is characterised in that：In the lysate
The guanidine hydrochloride can be 3-10M, can be 3.5-9.5M, can also be 5-9M；Can be further 5.5-7.5M；
And/or
The quality percent by volume of NaTDC can be 0.1-0.5% in the lysate, can be 0.2-0.4%, more
Can be 0.25-0.3%；And/or
The ionic surfactant is sodium N-lauroyl sarcosinate, and the quality percent by volume in the lysate is
0.8%-1.2%；And/or
The nonionic surface active agent is polyoxyethylene ether, and the quality concentration of volume percent in the lysate is
0.4%-0.5%.
5. the extracting method of bone marrow cell genomic DNA according to claim 3, it is characterised in that：First washing
Mixed liquor of the liquid for guanidine hydrochloride and absolute ethyl alcohol, pH value 9.0-10.0；
Optionally, in first cleaning solution guanidine hydrochloride molar concentration be more than the lysate in guanidine hydrochloride molar concentration；
And/or the volume ratio of guanidine hydrochloride and the absolute ethyl alcohol is 1 in first cleaning solution:(0.9‐1)；
Optionally, the concentration of guanidine hydrochloride is the concentration 1.4 of guanidine hydrochloride in the lysate again to 2 times in first cleaning solution.
6. the extracting method of bone marrow cell genomic DNA according to claim 3, it is characterised in that：Second washing
Mixed liquor of the liquid for Tris-HCl buffer solutions and absolute ethyl alcohol, pH value 7.5-8.5；
Preferably, in the Tris-HCl buffer solutions trishydroxymethylaminomethane (Tris) a concentration of 100 ± 10mM；And/or
The volume ratio of the Tris-HCl buffer solutions and absolute ethyl alcohol is 1:(3-7), or 1:(4‐5).
7. the extracting method of bone marrow cell genomic DNA according to claim 1, it is characterised in that：The eluent is
The aqueous solution of trishydroxymethylaminomethane (Tris) and ethylenediamine tetra-acetic acid (EDTA)；
Preferably, in the eluent trishydroxymethylaminomethane (Tris) a concentration of 10 ± 1mM；And/or the eluent
A concentration of 0.8-1mM of middle EDTA.
8. a kind of bone marrow cell genome DNA extracting reagent kit, it is characterised in that：It includes following reagent：Proteinase K reagents,
Lysate, ribonucleic acid enzymatic reagent, the first cleaning solution, the second cleaning solution, eluent and silica gel adsorption column.
9. bone marrow cell genome DNA extracting reagent kit according to claim 8, it is characterised in that：The protease examination
Agent contains the Proteinase K of 20 ± 10mg/mL；And/or
The lysate includes guanidine hydrochloride, NaTDC, ionic surfactant and nonionic surface active agent；With/
Or,
Mixed liquor of first cleaning solution for guanidine hydrochloride and absolute ethyl alcohol, pH value 9.0-10.0；And/or
Mixed liquor of second cleaning solution for Tris-HCl buffer solutions and absolute ethyl alcohol, pH value 7.5-8.5；And/or
The eluent is trishydroxymethylaminomethane (Tris) and the aqueous solution of ethylenediamine tetra-acetic acid (EDTA).
10. bone marrow cell genome DNA extracting reagent kit according to claim 9, it is characterised in that：In the lysate
The guanidine hydrochloride a concentration of 3-10M, further preferably preferably 3.5-9.5M, more preferably 5-9M, 5.5-7.5M；
And/or
In the lysate quality percent by volume of NaTDC be 0.1-0.5%, preferably 0.2-0.4%, more preferably
For 0.25-0.3%；And/or
The ionic surfactant is sodium N-lauroyl sarcosinate, and the quality percent by volume in the lysate is
0.8%-1.2%；And/or
The nonionic surface active agent is polyoxyethylene ether, and the quality percent by volume in the lysate is
0.4%-0.5%；And/or
Mixed liquor of first cleaning solution for guanidine hydrochloride and absolute ethyl alcohol, the concentration of guanidine hydrochloride is more than in first cleaning solution
The concentration of guanidine hydrochloride in the lysate；And/or
A concentration of 100 ± 10mM of trishydroxymethylaminomethane (Tris) in the Tris-HCl buffer solutions；And/or
A concentration of 10 ± 1mM of trishydroxymethylaminomethane (Tris) in the eluent,；And/or EDTA in the eluent
A concentration of 0.8-1mM.
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