CN103695413B - A kind of wall-breaking method of filamentous fungus extracting genome DNA - Google Patents
A kind of wall-breaking method of filamentous fungus extracting genome DNA Download PDFInfo
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- CN103695413B CN103695413B CN201310713074.5A CN201310713074A CN103695413B CN 103695413 B CN103695413 B CN 103695413B CN 201310713074 A CN201310713074 A CN 201310713074A CN 103695413 B CN103695413 B CN 103695413B
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Abstract
A wall-breaking method for filamentous fungus extracting genome DNA, adopts multiple centrifugation steps and glass stick extruding to carry out broken wall, specifically successively through fungus culture, collect thalline, multistep adds extracting solution, freezes, abolish thread thalline, collect Genomic DNA solution, the method for precipitation genomic dna.The method adopts the general refrigerator of-20 DEG C and homemade glass stick, experimental installation is easy to get, decrease the dependence to liquid nitrogen in traditional method, living contaminants during mortar grinder, and the method is easy to operate, economical and practical, filamentous fungus genomic dna can be obtained quickly and easily, can promote the use of.
Description
Technical field
The present invention relates to the wall-breaking method that a kind of genome extracts, the especially wall-breaking method of filamentous fungus genome leaching process necessity.
Background technology
Filamentous fungus is extensively present in occurring in nature, and has diversified function, and therefore the research of filamentous fungus is prolonged does not decline, and more and more causes the concern of investigator on the contrary.The first step of microbe research carries out species identification exactly, and the traditional classification appraisal basis of fungi mainly adopts morphological specificity during cultivation, and cultural characteristic difference highly significant, thus the accuracy of impact classification.The many-sides such as the modern classification qualification of fungi is then combining form, physiology and DNA molecular level carry out comprehensive identification.DNA extraction is the first step of molecular level qualification, about the method for filamentous fungus extracting genome DNA has a lot.Effectively extract the degree that the genomic key of filamentous fungus is to destroy filamentous fungal cells wall.Filamentous fungus genome DNA extracting method broadly similar, difference is mainly to adopt how to destroy filamentous fungal cells wall.The method of fungal cell's broken wall: 1) classical liquid nitrogen grinding method.Need special container to load the liquid nitrogen purchased, and grinding need more mycelium in mortar, simultaneously because the open area of mortar easily causes living contaminants comparatively greatly; The easy frostbite skin of liquid nitrogen, some local liquid nitrogen are not easy obtain and be restricted.2) frozen-thawed (with granulated glass sphere vibration) method.The method uses liquid nitrogen, adds cost and equipment requirements.3) enzymolysis process.For filamentous fungus broken wall enzyme costly, process a large amount of sample time very uneconomical.4) chemical reagent (Benzyl Chloride) method.There is the hidden danger in environmental protection in chemical reagent.
In order to obtain the general length consuming time of method that high quality DNA grows up, costly, and the equipment needing some special, general biology laboratory is difficult to meet its requirement.
Summary of the invention
The object of the present invention is to provide a kind of easy wall-breaking method for filamentous fungus extracting genome DNA, can the cell walls of broken filamentous fungus quickly and easily, adopt conventional centrifuge tube and self-control simple glass rod.The high-molecular-weight DNA that employing the method obtains, for analyses such as PCR, is applicable to carry out molecular biological research.
The present invention realizes above-mentioned purpose like this: a kind of wall-breaking method of filamentous fungus extracting genome DNA, and the method comprises the following steps:
1) filamentous fungus is inoculated in NBRIP (NationalBotanicalResearchInstitute ' sphosphatemedium), potato medium liquid substratum and cultivates;
2) filamentous fungus cultivated in step 1) is placed in centrifuge tube with sterile toothpick picking centrifugal, outwells supernatant liquor, collect thalline, then thalline is mixed centrifugal (12000rpm, 10min) after mycelia with sterilizing distilled water, abandon supernatant, retain hypha,hyphae;
3) in step 2) centrifugal after containing adding DNA extraction damping fluid in mycelium centrifuge tube, mixing thalline and Extraction buffer;
4) thalline that step 3) process obtains is put into-28 ~-18 DEG C of refrigerators immediately, place 1 ~ 2h and freeze;
5) thalline step 4) freezed, with the glass stick extrusion wall-breaking of sterilizing, repeating step 4) and step 5) 3 ~ 7 times (freezing-glass stick fragmentation), described glass stick holds end parcel 10-15 layer sterilized non-fat gauze, conveniently firmly, avoids the damage palm of the hand;
6) half of the bacterial cell disruption liquid obtained in step 5) is transferred in another centrifuge tube, then 65 DEG C of constant temperature 30min are all placed in, add isopyknic mixed liquor I (phenol: chloroform: primary isoamyl alcohol=25:24:1), fully mix, centrifugal (12000rpm, 10min), draw 600 ~ 700uL supernatant liquor to be moved in another centrifuge tube, add isopyknic mixed liquor I I (chloroform: primary isoamyl alcohol=24:1), fully mix, centrifugal (12000rpm, 10min);
7) supernatant liquor that step 6) obtains is moved in new centrifuge tube, and add the dehydrated alcohol of 2.5 times of volumes-20 DEG C of pre-freezes, centrifugal (12000rpm, 10min), abandon supernatant, the ethanol wash with 75% precipitates 2 times, obtains genomic dna and can be used for pcr amplification etc. after volatilizing, when color is difficult to remove colour substance with mixed solution very deeply, after being precipitated by genomic dna, extract test kit separation and purification with fungi again.
A kind of easy wall-breaking method for filamentous fungus extracting genome DNA provided by the invention, owing to adopting the general refrigerator of-20 DEG C and homemade glass stick, nearly all microbiology laboratory has configuration, avoids the demand of specific installation.Do not use liquid nitrogen can reduce the dependence of originating to liquid nitrogen, and living contaminants when decreasing mortar grinder.The method is easy to operate, economical and practical, can obtain filamentous fungus genomic dna quickly and easily, can promote the use of.
Embodiment
embodiment 1
For an easy wall-breaking method for filamentous fungus extracting genome DNA, the method comprises the following steps:
1) filamentous fungus cultivate: filamentous fungus A is incubated at NBRIP [NationalBotanicalResearchInstitute ' sphosphatemedium (pH7.0), often liter contains: 10gglucose, 5gCa
3(PO
4)
2, 5gMgCl
26H
2o, 0.25gMgSO
47H
2o, 0.2gKCl, 0.1g (NH
4)
2sO
4] in substratum, the Erlenmeyer flask of 250ml loads 50ml liquid nutrient medium, 28 DEG C of vibrations cultivations 2 days, then static gas wave refrigerator 4 days;
2) collect radicula byssoidea: the centrifuge tube centrifugal (12000rpm, 10min) being placed in 1.5ml with the fungi A cultivated in sterilizing toothpick picking step 1), outwell supernatant liquor, again with centrifugal (12000rpm after sterilizing distilled water mixing mycelia, 10min), abandon supernatant, retain hypha,hyphae;
3) extracting solution is added: add DNA extraction damping fluid 500ul [2%SDS, 1.4MNaCl, 0.2MTris-HCl (pH8.0), 0.02MEDTA (pH8.0)], and with sterile toothpick mixing thalline and Extraction buffer;
4) freeze: thalline step 3) process obtained puts into-20 DEG C of refrigerators immediately, place 2h liquid and freeze;
5) broken thread thalline: firmly by glass stick (the self-control diameter 0.5mm of sterilizing, length 11cm, hold end 10 layers of sterilized non-fat gauze parcel, damage palm of the hand when avoiding firmly) thalline in the centrifuge tube that freezed of extruding, 4 times repeatedly (freezing-glass stick attrition crushing);
6) Genomic DNA solution is collected: transfer in the centrifuge tube of another 1.5ml by the half of the bacterial cell disruption liquid obtained in step 5), two centrifuge tubes are placed in 65 DEG C of constant temperature 30min simultaneously, add isopyknic mixed liquor I (phenol: chloroform: primary isoamyl alcohol=25:24:1), abundant mixing, centrifugal (12000rpm, 10min), drawing 600uL supernatant liquor is moved in the centrifuge tube of same Zhi Xin, add isopyknic mixed liquor I I(chloroform: primary isoamyl alcohol=24:1), abundant mixing, centrifugal (12000rpm, 10min);
7) genomic dna is precipitated: supernatant liquor step 6) obtained is moved in new centrifuge tube, and adds the dehydrated alcohol of 2.5 times of volumes-20 DEG C of pre-freezes, centrifugal (12000rpm, 10min), remove supernatant liquor, the ethanol wash with 75% precipitates 2 times, obtains genomic dna after volatilizing;
8) order-checking and Molecular Identification: check order after the ITS sequence of genomic dna step 7) obtained carries out pcr amplification, obtain sequence map clearly and reach 520bp, find that this fungi belongs to by GenBank comparison
penicilliumbelong to, colonial morphology with
penicilliumbelong to and coincideing, called after
p.sp.XXR-A, the GenBank number of logging in: KF572125.
embodiment 2
For an easy wall-breaking method for filamentous fungus extracting genome DNA, the method comprises the following steps:
1) filamentous fungus cultivate: filamentous fungus B is incubated at NBRIP [NationalBotanicalResearchInstitute ' sphosphatemedium (pH7.0), often liter contains: 10gglucose, 5gCa
3(PO
4)
2, 5gMgCl
26H
2o, 0.25gMgSO
47H
2o, 0.2gKCl, 0.1g (NH
4)
2sO
4] in substratum, the Erlenmeyer flask of 250ml loads 50ml liquid nutrient medium, 28 DEG C of vibrations cultivations 2 days, then static gas wave refrigerator 6 days;
2) collect radicula byssoidea: the centrifuge tube centrifugal (12000rpm, 10min) being placed in 1.5ml with the fungi B cultivated in sterilizing toothpick picking step 1), outwell supernatant liquor, again with centrifugal (12000rpm after sterilizing distilled water mixing mycelia, 10min), abandon supernatant, retain hypha,hyphae;
3) extracting solution is added: add DNA extraction damping fluid 500ul [2%SDS, 1.4MNaCl, 0.2MTris-HCl (pH8.0), 0.02MEDTA (pH8.0)], and with sterile toothpick mixing thalline and Extraction buffer;
4) freeze: thalline step 3) process obtained puts into-28 DEG C of refrigerators immediately, place 2h liquid and freeze;
5) broken thread thalline: firmly by glass stick (the self-control diameter 0.5mm of sterilizing, length 12cm, hold end 13 layers of sterilized non-fat gauze parcel, damage palm of the hand when avoiding firmly) thalline in the centrifuge tube that freezed of extruding, 5 times repeatedly (freezing-glass stick attrition crushing);
6) Genomic DNA solution is collected: transfer in the centrifuge tube of another 1.5ml by the half of the bacterial cell disruption liquid obtained in step 5), two centrifuge tubes are placed in 65 DEG C of constant temperature 30min simultaneously, add isopyknic mixed liquor I (phenol: chloroform: primary isoamyl alcohol=25:24:1), abundant mixing, centrifugal (12000rpm, 10min), drawing 700uL supernatant liquor is moved in the centrifuge tube of same Zhi Xin, add isopyknic mixed liquor I I(chloroform: primary isoamyl alcohol=24:1), abundant mixing, centrifugal (12000rpm, 10min);
7) genomic dna is precipitated: supernatant liquor step 6) obtained is moved in new centrifuge tube, and adds the dehydrated alcohol of 2.5 times of volumes-20 DEG C of pre-freezes, centrifugal (12000rpm, 10min), remove supernatant liquor, the ethanol wash with 75% precipitates 2 times, obtains genomic dna after volatilizing;
8) fungal genomic DNA extraction test kit is removed variegated: the genomic dna precipitation that step 7) obtains, containing darker black impurity, extracts test kit (BiospinfungusgenomicDNAextractionKit) purifying again with fungal genomic DNA;
9) order-checking and Molecular Identification (validity of broken wall): check order after the ITS sequence of genomic dna step 8) obtained carries out pcr amplification, obtain sequence map clearly and reach more than 560bp, find that this fungi belongs to by GenBank comparison
aspergillusbelong to, colonial morphology with
aspergillusbelong to and coincideing, called after
a.sp.XXR-B, the GenBank number of logging in: KF572126.
embodiment 3
For an easy wall-breaking method for filamentous fungus extracting genome DNA, the method comprises the following steps:
1) filamentous fungus is cultivated: be incubated at by filamentous fungus C in potato substratum (20% potato, 2% sucrose), and the Erlenmeyer flask of 250ml loads 50ml liquid nutrient medium, 28 DEG C of vibrations cultivations 1 day, then static gas wave refrigerator 6 days;
2) collect radicula byssoidea: the centrifuge tube centrifugal (12000rpm, 10min) being placed in 1.5ml with the fungi C cultivated in sterilizing toothpick picking step 1), outwell supernatant liquor, again with centrifugal (12000rpm after sterilizing distilled water mixing mycelia, 10min), abandon supernatant, retain hypha,hyphae;
3) extracting solution is added: add DNA extraction damping fluid 500ul [2%SDS, 1.4MNaCl, 0.2MTris-HCl (pH8.0), 0.02MEDTA (pH8.0)], and with sterile toothpick mixing thalline and Extraction buffer;
4) freeze: thalline step 3) process obtained puts into-20 DEG C of refrigerators immediately, place 2h liquid and freeze;
5) broken thread thalline: firmly by glass stick (the self-control diameter 0.4mm of sterilizing, length 12cm, hold end 12 layers of sterilized non-fat gauze parcel, damage palm of the hand when avoiding firmly) thalline in the centrifuge tube that freezed of extruding, 4 times repeatedly (freezing-glass stick attrition crushing);
6) Genomic DNA solution is collected: transfer in the centrifuge tube of another 1.5ml by the half of the bacterial cell disruption liquid obtained in step 5), two centrifuge tubes are placed in 65 DEG C of constant temperature 30min simultaneously, add isopyknic mixed liquor I (phenol: chloroform: primary isoamyl alcohol=25:24:1), abundant mixing, centrifugal (12000rpm, 10min), drawing 600uL supernatant liquor is moved in the centrifuge tube of same Zhi Xin, add isopyknic mixed liquor I I(chloroform: primary isoamyl alcohol=24:1), abundant mixing, centrifugal (12000rpm, 10min);
7) genomic dna is precipitated: supernatant liquor step 6) obtained is moved in new centrifuge tube, and adds the dehydrated alcohol of 2.5 times of volumes-20 DEG C of pre-freezes, centrifugal (12000rpm, 10min), remove supernatant liquor, the ethanol wash with 75% precipitates 2 times, obtains genomic dna after volatilizing;
8) order-checking and Molecular Identification: check order after the ITS sequence of genomic dna step 7) obtained carries out pcr amplification, obtain sequence map clearly and reach more than 580bp, find that this fungi belongs to by GenBank comparison
trichodermabelong to, colonial morphology with
trichodermabelong to and coincideing, called after
t.sp.H09, the GenBank number of logging in: HM000038.
Claims (7)
1. a wall-breaking method for filamentous fungus extracting genome DNA, comprises the following steps,
1) cultivate: filamentous fungus is inoculated in liquid nutrient medium, under 25 ~ 30 DEG C of environment, vibrations cultivation 1 ~ 2 day, static gas wave refrigerator 2 ~ 7 days;
2) collect thalline: it is centrifugal that picking filamentous fungus mycelia is placed in centrifuge tube, outwells supernatant liquor, collect thalline, then thalline is mixed after mycelia centrifugal with sterilizing distilled water, abandon supernatant, retain hypha,hyphae;
3) add extracting solution: in step 2) centrifugal after containing adding DNA extraction damping fluid in mycelium centrifuge tube, mixing thalline and Extraction buffer, described DNA extraction damping fluid is 2%SDS, 1.4MNaCl, 0.2MTris-HCl, the mixed solution of 0.02MEDTA, wherein 0.2MTris-HClpH is 8.0,0.02MEDTApH is 8.0;
4) freeze: the thalline that step 3) process obtains is put into-28 ~-18 DEG C of refrigerators, place 1 ~ 2h and freeze;
5) broken thread thalline: the thalline that step 4) has been freezed, with the glass stick extrusion wall-breaking of sterilizing, described extrusion wall-breaking firmly the glass stick of sterilizing is extruded the thalline in the centrifuge tube that freezed, 5 times repeatedly, freeze-glass stick attrition crushing, thus realize the effect of thalline extraction DNA;
6) collect Genomic DNA solution: repeating step 4) and step 5) 3 ~ 7 times after, the half of the bacterial cell disruption liquid obtained after broken wall is transferred in another centrifuge tube, be placed in 65 DEG C of constant temperature 30min, add isopyknic mixed liquor I, fully mix, centrifugal, drawing 600 ~ 700uL supernatant liquor is moved in the centrifuge tube of same, add isopyknic mixed liquor I I simultaneously, fully mix, centrifugal;
7) genomic dna is precipitated: supernatant liquor step 6) obtained is moved in new centrifuge tube, and add the dehydrated alcohol of 2.5 times of volumes-20 DEG C of pre-freezes, and centrifugal, remove supernatant liquor, ethanol wash with 75% precipitates 2 times, obtains filamentous fungus genomic dna after volatilizing.
2. the wall-breaking method of filamentous fungus extracting genome DNA according to claim 1, it is characterized in that: in step 1), described liquid nutrient medium is NBRIP (NationalBotanicalResearchInstitute ' sphosphatemedium) or potato substratum.
3. the wall-breaking method of filamentous fungus extracting genome DNA according to claim 2, is characterized in that: described NBRIP substratum often rises and contains: 10gC
6h
12o
6, 5gCa
3(PO
4)
2, 5gMgCl
26H
2o, 0.25gMgSO
47H
2o, 0.2gKCl, 0.1g (NH
4)
2sO
4, medium pH is 7.0 ~ 7.2; Described potato substratum contains 20% potato, 2% sucrose.
4. the wall-breaking method of filamentous fungus extracting genome DNA according to claim 1, is characterized in that: step 2) in, by the mycelium distilled water rinsing 1 ~ 2 time of collecting, to remove the meta-bolites of substratum and fungi.
5. the wall-breaking method of filamentous fungus extracting genome DNA according to claim 1, is characterized in that: glass stick sterilizing in step 5), holds end parcel 10 ~ 15 layers of sterilized non-fat gauze.
6. the wall-breaking method of filamentous fungus extracting genome DNA according to claim 1, is characterized in that: in step 6), and mixed liquor I is V
phenol: V
chloroform: V
primary isoamyl alcohol=25:24:1; Mixed liquor I I is V
chloroform: V
primary isoamyl alcohol=24:1.
7. the wall-breaking method of filamentous fungus extracting genome DNA according to claim 1, is characterized in that: described centrifugation rate is 12000rpm, and centrifugation time is 10min.
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| CN106947758B (en) * | 2017-02-24 | 2020-06-26 | 许昌学院 | Technology for rapidly extracting filamentous fungus DNA (deoxyribonucleic acid) by PCR (polymerase chain reaction) amplification |
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| 一种高效提取真菌总DNA 的方法;孙立夫 等;《菌物学报》;20090315;第28卷(第2期);第300页第1.2节，图1 * |
| 冻融法快速提取真菌微量培养物基因组ＤＮＡ;曾东方 等;《生物技术》;20101231;第20卷(第6期);第49-51页 * |
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