CN106754877A - The extracting method of transgene rape Bee Pollen DNA - Google Patents
The extracting method of transgene rape Bee Pollen DNA Download PDFInfo
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- CN106754877A CN106754877A CN201611145606.XA CN201611145606A CN106754877A CN 106754877 A CN106754877 A CN 106754877A CN 201611145606 A CN201611145606 A CN 201611145606A CN 106754877 A CN106754877 A CN 106754877A
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Abstract
The invention discloses a kind of extracting method of transgene rape Bee Pollen DNA, micro Bee Pollen nitrogen is ground into broken wall first；Secondly high salt Extraction buffer, SDS and Proteinase K are added；Then 65 DEG C of h of water-bath 2, add appropriate NaCl solution, and 10000 rpm are centrifuged 30 min, take supernatant；Then 20 DEG C of isopropanols of precooling of certain volume, 20 DEG C of 2 h of placement are added in supernatant；Last 10000 rpm is centrifuged 10 min and abandons supernatant, and sediment is washed 2 ~ 3 times with the ethanol that mass concentration is 75%, removes ethanol natural air drying, obtains final product bee pollen form cole nucleic acid.The beneficial effect of transgene rape Bee Pollen DNA extraction method of the invention is：Extract the high-quality DNA for being suitable for PCR detections from transgene rape Bee Pollen, simple to operate, low toxicity, it is time-consuming it is short, beneficial to quick detection.
Description
Technical field
The invention belongs to biological technical field, and in particular to the extracting method of transgene rape Bee Pollen DNA.
Background technology
Rape (Brassica napus L. it is) one of big oil crops in the world four, while being also important nectar source crop.
Rape pollen is that honeybee gathers pollen grain from rape pistil, and adds special glandular secretion thing（Nectar and saliva）Mixing and
Into a kind of irregular discoid shape thing；It is rich in protein, polysaccharide, vitamin, trace element, unrighted acid etc., is
A kind of nutritional health food of high protein and low fat.There are some researches show bee pollen form cole has antifatigue, enhancing human immunity
The multiple efficacies such as function, protection cardio-cerebrovascular, improvement incretory secreting function, protection liver.
With the maturation of transgenic technology, the genetically modified crops of commercial growth are all continuously increased in species and area,
Transgene rape large area commercial growth already abroad.China commercially produces there is presently no transgene rape, but
Genetic transformation and its field test about transgene rape is very active.Pollen is that the arrenotoky in flowering plant stamen is thin
Born of the same parents, it carries the hereditary information of life, also necessarily includes transgene component.Rape pollen is a kind of maximum pollen of yield,
In addition to as health products direct marketing, also frequently as pharmaceutical raw material.Therefore, the detection work to foreign gene in rape pollen is compeled
In the eyebrows and eyelashes, the good and enough transgene rape Bee Pollen DNA of quality how is disposably obtained, be the premise that related work is carried out.
The content of the invention
The purpose of the present invention is：A kind of extracting method of transgene rape Bee Pollen DNA is provided, can be divided using the method
The transgene rape Bee Pollen DNA that purity is high, integrality is good is separated out, simple to operate beneficial to PCR quick detections, low toxicity takes
It is short.
Technical solution of the invention is：Bee Pollen is ground into broken wall；Add high salt Extraction buffer, SDS and albumen
Enzyme K, mixes, and is put into 65 DEG C of water-bath 1.5-2.5 h；NaCl solution is added, is mixed, 10000 rpm centrifugation 20-30 min take
Supernatant；- 20 DEG C of isopropanols of precooling, -20 DEG C of 1.5 ~ 2.5 h of placement are added in supernatant；10000 rpm are centrifuged 5-10
Min abandons supernatant, and sediment is washed 2 ~ 3 times with the ethanol of mass concentration 75%, removes ethanol natural air drying, obtains final product bee pollen form cole core
Acid.
Wherein, described transgene rape Bee Pollen is liquid nitrogen cryogenics grinding broken wall treatment, and its condition is：By transgenosis
To 60-100 mesh, liquid nitrogen volatility is extremely strong, and whole process of lapping need to constantly add liquid nitrogen, make sample for bee pollen form cole cryogrinding
Leaching is in the sample.
Wherein, in described high salt Extraction buffer component include 0.4 mol/L NaCl, 0.1 mol/L Tris-HCl,
0.02 mol/L EDTA, 1%SDS, pH scopes 7.7-8.3.
Wherein, the extracting method of transgene rape Bee Pollen DNA includes operating procedure in detail below：
（1）Bee Pollen 0.08-0.12 g are placed in mortar, it is powdered that liquid feeding nitrogen grinds to form 60-100 mesh, add 2 ml centrifugations
Guan Zhong；
（2）400 μ L high salts Extraction buffers, 20 mg/ of the SDS of 40 μ L mass concentrations 20% and 8 μ L are added in centrifuge tube
Ml Proteinase Ks, fully mix, and are put into 65 DEG C of water-bath 1.5-2.5 h, and period turns upside down mixing every 20 min；
（3）Centrifuge tube is taken out, room temperature is cooled to, the 6 mol/L NaCl of 300 μ L are added, is vortexed with the rpm of rotating speed 2500 and mixed
20-30 s, 10000 rpm centrifugation 20-30 min, take supernatant；
（4）Addition is the isopropanol of its -20 DEG C of precooling of 2/3 volume, -20 DEG C of 2 ~ 3 h of placement in supernatant；
（5）10000 rpm centrifugations 5-10 min abandon supernatant, and sediment is washed 2 ~ 3 times with the ethanol of mass concentration 75%, removes ethanol
Natural air drying, obtains final product bee pollen form cole nucleic acid；
（6）Bee pollen form cole nucleic acid is dissolved in 50-100 μ L sterilized waters, in 0 ~ 4 DEG C of preservation, or long-term guarantor is placed in -20 DEG C
Deposit.
The present invention has the advantages that：
1st, pollen wall is destroyed using the method for physical low-temperature, pollen content is discharged, instead of the use of lysate, reduces cost.
2nd, current methods there is a problem of using a large amount of toxic reagents, such as chloroform, beta -mercaptoethanol, isoamyl alcohol and saturation
Phenol, such reagent is poisonous and sends niff, and risk is brought to experimental implementation person, and easily pollutes environment, and this
Inventive method avoids using a large amount of toxic reagents.
3rd, the present invention extracts the high-quality DNA for being suitable for PCR detections, operation letter from transgene rape Bee Pollen
It is single, low toxicity, time-consuming short, recovery rate is high, and beneficial to quick detection, 0.1 g bee pollen form coles extract 80 μ L sterilized waters of gained nucleic acid
Dissolving can be directly used for regular-PCR detection, the need for meeting the detection of low content foreign gene.
Brief description of the drawings
Fig. 1 is bee pollen form cole STb gene electrophoretic band.
Fig. 2 is the endogenous PEP genes electrophoretic band of bee pollen form cole rape.
Fig. 3 is transgene rape Bee Pollen foreign gene Bar gene electrophoretic bands.
Specific embodiment
Technical scheme is described in detail with reference to embodiment, it should be understood that protection scope of the present invention
Do not limited by specific embodiment.Explicitly indicated that unless otherwise other, otherwise in entire disclosure and claims, art
Language " including " or its conversion such as "comprising" or " including " etc. will be understood to comprise stated element or part, and
Other elements or other parts are not excluded.
Embodiment 1：3 parts of ordinary cole Bee Pollen is chosen, following extractions are carried out using the method for the present invention：
（1）0.08 g Bee Pollen liquid feeding nitrogen is ground to form into 60 mesh powdered, in adding 2 ml centrifuge tubes；
（2）400 μ L high salts Extraction buffers, the SDS of 40 μ L mass concentrations 20%, 20 mg/ of 8 μ L are added in centrifuge tube
Ml Proteinase Ks, are fully mixed with refiner with 2500 rpm, are put into 65 DEG C of h of water-bath 1.5, and period turns upside down every 20 min
Mix；
（3）Centrifuge tube is taken out, room temperature is cooled to, the mol/L NaCl of 300 μ L 6 are added, is vortexed with 2500 rpm and is mixed 20 s,
10000 rpm are centrifuged 30 min, take supernatant；
（4）It is the isopropanol of its addition -20 DEG C of precooling of 2/3 volume, -20 DEG C of 2 h of placement in supernatant；
（5）10000 rpm are centrifuged 8 min and abandon supernatant, and sediment is washed 2 times with the ethanol of mass concentration 75%, removes ethanol natural wind
It is dry, obtain final product bee pollen form cole nucleic acid；
（6）Bee pollen form cole nucleic acid is dissolved in 50 μ L sterilized waters, in 0 ~ 4 DEG C of preservation.
First, the bee pollen form cole STb gene that embodiment 1 is extracted is detected with agarose gel electrophoresis method, as shown in figure 1, to 3 parts
Ordinary cole Bee Pollen sample detects clearly electrophoretic band, illustrates that this method is adapted to the extraction of bee pollen form cole STb gene.
2nd, the quality and yield of the rape flower Bee Pollen STb gene that spectrophotometer method detection embodiment 1 is extracted, use
Eppendorf Biophotometer protein nucleic acid analyzers detect its quality and yield, as a result see the table below：
As seen from the above table, the impurity such as albumen, polysaccharide, polyphenol is less in the DNA that the inventive method is extracted, and DNA purity is high, therefore this
Inventive method is applied to the extraction of bee pollen form cole STb gene.
Embodiment 2：3 parts of ordinary cole Bee Pollen is chosen, following extractions are carried out using the inventive method：
（1）0.1 g Bee Pollen liquid feeding nitrogen is ground to form into 80 mesh powdered, in adding 2 ml centrifuge tubes；
（2）400 μ L high salts Extraction buffers, the SDS of 40 μ L 20%, the mg/ml Proteinase Ks of 8 μ L 20 are added in centrifuge tube,
Fully mixed with 2500 rpm with refiner, be put into 65 DEG C of h of water-bath 2, period turns upside down mixing every 20 min；
（3）Centrifuge tube is taken out, room temperature is cooled to, the mol/L NaCl of 300 μ L 6 are added, is vortexed with 2500 rpm and is mixed 25 s,
10000 rpm are centrifuged 30 min, take supernatant；
（4）Addition is the isopropanol of its -20 DEG C of precooling of 2/3 volume in supernatant.- 20 DEG C of 2.5 h of placement；
（5）10000 rpm are centrifuged 8 min and abandon supernatant, and sediment is washed 2 times with the ethanol of mass concentration 75%, removes ethanol natural wind
It is dry, obtain final product bee pollen form cole nucleic acid；
（6）Bee pollen form cole nucleic acid is dissolved in 80 μ L sterilized waters, in 0 ~ 4 DEG C of preservation, for PCR detections.
The PCR detections of rape endogenous gene PEP genes：Amplified fragments 248bp long, forward primer：
GCTAGTGTAGACCAGTTCTTG, reverse primer：CACTCTTGTCTCTTGTCCTC；PCR reaction systems：0.5 U Taq enzymes,
2.5 25 mmol/L MgCl of μ L 10 × Buffer, 2 μ L2, 0.25 μ L 10 mmol/L dNTPs, 0.5 μ L 10 μ
Mol/L Primers, 2.5 μ L template DNAs；Reaction volume is 25 μ l；Amplification condition is：94 DEG C of predegeneration 2min；94 DEG C of denaturation
30s, 60 DEG C of annealing 30s, 72 DEG C of 30 s of extension, 30 circulations；72 DEG C of 10 min of extension, 4 DEG C of preservations；PCR primer carries out 1% fine jade
Sepharose electrophoretic analysis.
Result detection is as follows：
First, the PEP for obtaining is expanded with PCR in the bee pollen form cole STb gene of the extraction of agarose gel electrophoresis method detection embodiment 2
Gene, as shown in Fig. 2 detecting PEP genes clearly electrophoretic band to 3 parts of bee pollen form cole samples, illustrates that this method is carried
The bee pollen form cole STb gene for taking can be expanded to rape endogenous gene PEP genes.
Embodiment 3：3 parts of transgene rape Bee Pollen is chosen, following extractions are carried out using the inventive method：
（1）0.12 g Bee Pollen liquid feeding nitrogen is ground to form into 100 mesh powdered, in adding 2 ml centrifuge tubes；
（2）400 μ L high salts Extraction buffers, the SDS of 40 μ L 20%, the mg/ml Proteinase Ks of 8 μ L 20 are added in centrifuge tube,
Fully mixed with 2500 rpm with refiner, be put into 65 DEG C of h of water-bath 2.5, period turns upside down mixing every 20 min；
（3）Centrifuge tube is taken out, room temperature is cooled to, the mol/L NaCl of 300 μ L 6 are added, is vortexed with 2500 rpm and is mixed 30 s,
10000 rpm are centrifuged 30 min, take supernatant；
（4）Addition is the isopropanol of its -20 DEG C of precooling of 2/3 volume in supernatant.- 20 DEG C of 3 h of placement；
（5）10000 rpm are centrifuged 8 min and abandon supernatant, and sediment is washed 3 times with the ethanol of mass concentration 75%, removes ethanol natural wind
It is dry, obtain final product bee pollen form cole nucleic acid；
（6）Bee pollen form cole nucleic acid is dissolved in 100 μ L sterilized waters, in 0 ~ 4 DEG C of preservation, for PCR detections.
The PCR detections of transgene rape external source base Bar genes：Amplified fragments 595bp long, forward primer：
CCTTCGCAAGACCCTTCCTC, reverse primer：AAACCCACGTCATGCCAGTT；PCR reaction systems：0.5 U Taq enzymes,
2.5 25 mmol/L MgCl of μ L 10 × Buffer, 2 μ L2, 0.5 μ L 10 mmol/L dNTPs, 0.5 μ L 10 μ
Mol/L Primers, 2.5 μ L template DNAs；Reaction volume is 25 μ L；Amplification condition is：94 DEG C of predegeneration 2min；94 DEG C of denaturation
30s, 60 DEG C of annealing 30s, 72 DEG C of 30 s of extension, 30 circulations；72 DEG C of 10 min of extension, 4 DEG C of preservations；PCR primer carries out 1% fine jade
Sepharose electrophoretic analysis.
Result detection is as follows：
First, expanded with PCR in the transgene rape Bee Pollen STb gene of the extraction of agarose gel electrophoresis method detection embodiment 3
The Bar genes for arriving, as shown in figure 3, Bar genes clearly electrophoretic band is detected to 3 parts of transgene rape Bee Pollen samples,
Illustrate that this method is fitted the bee pollen form cole STb gene of extraction and can be expanded to rape foreign gene Bar genes.
Claims (4)
1. the extracting method of transgene rape Bee Pollen DNA, it is characterised in that：Bee Pollen is ground into broken wall；High salt is added to extract
Buffer solution, SDS and Proteinase K, mix, and are put into 65 DEG C of water-bath 1.5-2.5 h；NaCl solution is added, is mixed, 10000 rpm
Centrifugation 20-30 min, take supernatant；- 20 DEG C of isopropanols of precooling, -20 DEG C of 1.5 ~ 2.5 h of placement are added in supernatant；
10000 rpm centrifugations 5-10 min abandon supernatant, and sediment is washed 2 ~ 3 times with the ethanol of mass concentration 75%, removes ethanol natural wind
It is dry, obtain final product bee pollen form cole nucleic acid.
2. the extracting method of transgene rape Bee Pollen DNA according to claim 1, it is characterised in that：Described turns base
Because bee pollen form cole is liquid nitrogen cryogenics grinding broken wall treatment, its condition is：By transgene rape Bee Pollen cryogrinding to 60-
100 mesh, whole process of lapping need to constantly add liquid nitrogen, sample is soaked in the sample.
3. method according to claim 1, it is characterised in that：Component includes 0.4 mol/L in high salt Extraction buffer
NaCl, 0.1 mol/L Tris-HCl, 0.02 mol/L EDTA, 1%SDS, pH scopes 7.7-8.3.
4. the extracting method of transgene rape Bee Pollen DNA according to claim 1, it is characterised in that：The transgenic rape
The extracting method of dish Bee Pollen DNA includes operating procedure in detail below：
（1）Bee Pollen 0.08-0.12 g are placed in mortar, it is powdered that liquid feeding nitrogen grinds to form 60-100 mesh, add 2 ml centrifugations
Guan Zhong；
（2）400 μ L high salts Extraction buffers, 20 mg/ of the SDS of 40 μ L mass concentrations 20% and 8 μ L are added in centrifuge tube
Ml Proteinase Ks, fully mix, and are put into 65 DEG C of water-bath 1.5-2.5 h, and period turns upside down mixing every 20 min；
（3）Centrifuge tube is taken out, room temperature is cooled to, the 6 mol/L NaCl of 300 μ L are added, is vortexed with the rpm of rotating speed 2500 and mixed
20-30 s, 10000 rpm centrifugation 20-30 min, take supernatant；
（4）Addition is the isopropanol of its -20 DEG C of precooling of 2/3 volume, -20 DEG C of 2 ~ 3 h of placement in supernatant；
（5）10000 rpm centrifugations 5-10 min abandon supernatant, and sediment is washed 2 ~ 3 times with the ethanol of mass concentration 75%, removes ethanol
Natural air drying, obtains final product bee pollen form cole nucleic acid；
（6）Bee pollen form cole nucleic acid is dissolved in 50-100 μ L sterilized waters, in 0 ~ 4 DEG C of preservation, or long-term guarantor is placed in -20 DEG C
Deposit.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611145606.XA CN106754877A (en) | 2016-12-13 | 2016-12-13 | The extracting method of transgene rape Bee Pollen DNA |
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| CN201611145606.XA CN106754877A (en) | 2016-12-13 | 2016-12-13 | The extracting method of transgene rape Bee Pollen DNA |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113854200A (en) * | 2021-08-26 | 2021-12-31 | 中国农业科学院油料作物研究所 | Method for collecting transgenic rape pollen to be screened in large scale by utilizing bees |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101117634A (en) * | 2007-07-09 | 2008-02-06 | 华中农业大学 | Method for separating cotton chloroplast DNA |
| CN102796731A (en) * | 2012-08-21 | 2012-11-28 | 中国科学院西北高原生物研究所 | Method for extracting nucleic acid of rape bee pollen |
| CN103146682A (en) * | 2012-11-30 | 2013-06-12 | 青海睿元药物研究所有限责任公司 | Extraction method of bee pollen nucleinic acid |
-
2016
- 2016-12-13 CN CN201611145606.XA patent/CN106754877A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101117634A (en) * | 2007-07-09 | 2008-02-06 | 华中农业大学 | Method for separating cotton chloroplast DNA |
| CN102796731A (en) * | 2012-08-21 | 2012-11-28 | 中国科学院西北高原生物研究所 | Method for extracting nucleic acid of rape bee pollen |
| CN103146682A (en) * | 2012-11-30 | 2013-06-12 | 青海睿元药物研究所有限责任公司 | Extraction method of bee pollen nucleinic acid |
Non-Patent Citations (2)
| Title |
|---|
| 张云贵 等: "《生物化学实验指导》", 31 December 2007, 中国农业出版社 * |
| 钱鑫萍 等: "《生物化学实验指导书》", 31 July 2016, 合肥工业大学出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113854200A (en) * | 2021-08-26 | 2021-12-31 | 中国农业科学院油料作物研究所 | Method for collecting transgenic rape pollen to be screened in large scale by utilizing bees |
| CN113854200B (en) * | 2021-08-26 | 2023-10-03 | 中国农业科学院油料作物研究所 | Method for collecting transgenic rape pollen to be screened on large scale by using bees |
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