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1、第六章 基因组dna的提取（The sixth chapter is the extraction of genomic DNA）The sixth chapter is the extraction of genomic DNAThe first section is an overviewGenomic DNA extraction is usually used to construct genomic libraries, Southern hybrids (including RFLP) and PCR segregating genes. With the longer natur
2、e of genomic DNA, it can be separated from small molecules such as organelles or plasmids, DNA. Adding a certain amount of isopropanol or ethanol, high molecular DNA genome or precipitation form fibrous floc floating the available glass rod to remove it, and small molecule DNA can only form granular
3、 sediment attached to wall and bottom, so as to achieve the purpose of extraction. In the process of extraction, the chromosome will occur mechanical faults, produce fragments of different size, should be operated under mild conditions so isolating genomic DNA, such as minimizing phenol / chloroform
4、 extraction, blending process should be gentle, to get longer DNA. In general, the genomic DNA must be more than 100kb in the construction of genomic libraries. Otherwise, very few fragments with suitable ends are removed on both sides of the enzyme. For RFLP and PCR analysis, the length of DNA can
5、be as short as 50kb, and above this length, RFLP fragments (below 20KB) can be guaranteed after enzyme digestion, and the fragments amplified by PCR (below 2KB) can be guaranteed.Different biological (plant, animal and microorganism) have different extraction methods of genomic DNA; different tissue
6、s of different species or the same species due to different cell structure and composition of the separation methods are also different. When extracting the DNA of a particular organization, the corresponding extraction method must be established by referring to the literature and experience to obta
7、in the available DNA macromolecules. In particular, polysaccharides and enzymes in tissues have a strong inhibitory effect on subsequent enzymatic digestion and PCR reaction. Therefore, polysaccharides and phenols should be considered when genomic DNA is extracted from materials rich in these substa
8、nces.In this study, we used rice seedlings (He Benke), Li (Apple) leaves, animal muscle tissue and Escherichia coli culture as materials to study the general method of genomic DNA extraction.In the second section, genomic DNA was extracted from plant tissuesI. materialThe young leaves of a young pla
9、nt or other plant of the family gramineae.Two, equipmentPipette, refrigeration centrifuge, centrifuge table high speed, water bath, ceramic mortar, 50ml centrifugal tube (with cover) and 5ml and 1.5ml tube bent into a hook shaped small glass rod.Three reagents1, extract buffer I: 100mmol/L, Tris, Cl
10、, pH8.0, 20mmol/L, EDTA, 500mmol/L, NaCl, 1.5% SDS.2, extract buffer II: 18.6g glucose, 6.9g two, ethyl two, sodium thiosulfate, 6.0gPVP, 240ul, mercapto ethanol, add water to 300ml.3, 80:4:16/ chloroform: amyl alcohol: Ethanol4, RnaseA mother liquor: see Chapter 1 for recipes.5, other reagents: liq
11、uid nitrogen, isopropyl alcohol, TE buffer, anhydrous ethanol, 70% ethanol, 3mol/L, NaAc.Four, operation steps:(a) extracted from rice seedlings or other grass DNA1. 20ml buffer was added into the 50ml centrifuge tube and I water bath was preheated at 60 DEG C.2. rice seedlings or leaves 5-10g, minc
12、ed and powdered in a mortar with liquid nitrogen immediately after preheating into the centrifuge tube, shaking and mixing, 60 DEG C water bath for 30-60 minutes (a long time, the high yield of DNA), shaking from time to time.3. add 20ml chloroform / amyl alcohol / ethanol solution, mix it upside do
13、wn (with gloves to prevent damage to the skin), leave it at room temperature for 5-10 minutes, so that the aqueous phase and organic phase are layered (if necessary, can be mixed).4. 5000rpm centrifugation at room temperature for 5 minutes.5., carefully remove the supernatant to another 50ml centrif
14、uge tube, adding 1 times the volume of isopropanol, mixed, placed at room temperature for a moment, that occurs floc DNA precipitation.6. add 1ml TE in 1.5ml eppendorf. Use a hooked glass rod to remove DNA floc, blot it on a clean absorbent paper and transfer it into a centrifuge tube containing TE.
15、 DNA dissolves rapidly at TE.7., such as DNA does not form floc precipitation,5000rpm can be centrifuged for 5 minutes, and then the precipitation into the TE tube. Such precipitation is often difficult to dissolve in TE, can be placed in the water bath at 60 degrees for more than 15 minutes to help
16、 dissolve.8. centrifuge DNA solution 3000rpm for 5 minutes and pour the supernatant into a clean 5ml centrifuge tube.9. add 5 mu L RNaseA (10 g/ L), 37 degrees 10 minutes, remove RNA (RNA operation on DNA, analysis general no influence, can omit this step).10. add 1/10 volume of 3mol/L, NaAc and 2 x
17、 volume of ice ethanol, mix, place at -20 DEG C for about 20 minutes, and DNA form flocculent precipitate.11. remove DNA deposits with a glass stick, rinse with 70% ethanol, and then dry on a clean absorbent paper.12. re dissolve DNA in 1ml, TE, -20 storage.13. samples of 2 L DNA were collected on 0
18、.7% Agarose gel, and the molecular size of DNA was detected. At the same time, take 15 l dilution 20 times, determine OD260/OD280, detect the content and quality of DNA.note 5g samples are guaranteed to be 500 g DNA, sufficient for RFLP, PCR and other analysis purposes.(two) genomic DNA was extracte
19、d from plum (Apple) leaves1., take 3-5 grams of tender leaves, liquid nitrogen into powder.2. add extraction buffer, II 10ml, and then grinding to dissolve pulp. 10000rpm, 10min.3., the supernatant, precipitation and extraction liquid I 20ml, mixing. 65 degrees, 30-60min, shake frequently.4. operate
20、 with step 3-13 in this section (1). ?In the third section, genomic DNA was extracted from animal tissuesI. materialMammalian fresh tissue.Two, equipmentPipette, high speed refrigerated centrifuge, table centrifuge, water bath pot.Three reagents1, separation buffer: 10mmol/L, Tris, Cl, pH7.4, 10mmol
21、/L, NaCl, 25mmol/L, EDTA.2, other reagents: 10% SDS, protease K (20mg/ml or powder), ether, phenol: chloroform: isoamyl alcohol (25:24:1), anhydrous ethanol and 70% ethanol, 5mol/L, NaCl, 3mol/L, NaAc, TE.Four, operation steps:1. cut tissue around 5g, excluding the connective tissue, blood sucking a
22、bsorbent paper, cut into a mortar (the better).2. pour the liquid nitrogen into the powder and add 10ml to separate the buffer.3. add 1ml 10% SDS, mix evenly, at this point the sample becomes very viscous.4. add 50ul or 1mg protease K, keep warm for 1-2 hours at 37 hours, until the tissue is complet
23、ely disintegrated.5. add 1ml, 5mol/L, NaCl, mix, 5000rpm, centrifugal for a few seconds.6. take the supernatant in the new centrifuge tube and extract with the equal volume phenol: chloroform: isoamyl alcohol (25:24:1). After stratification, the 3000rpm is centrifuged for 5 minutes.7. take the water
24、 from the upper layer to the clean centrifuge tube and extract with twice the volume of ether (operate under the condition of ventilation).8. remove the upper ether and retain the lower aqueous phase.9. plus 1/10, volume 3mol/L, NaAc, and 2 times the volume of anhydrous ethanol, reverse mixing preci
25、pitation DNA. At room temperature for 10-20 min, DNA precipitates to form white floc.10., with a stick hook out of DNA precipitation, 70% ethanol rinse, absorbent paper on the dry, dissolved in 1ml, TE, -20 degrees preservation.11. if there is insoluble particles in DNA solution, can be briefly cent
26、rifuged in 5000rpm, Torikami Kiyo; if you want to remove the RNA, you can add 5 mu L RNaseA (10 g/ L), and keep 37 minutes for 30 minutes. After phenol extraction, precipitate DNA according to step 9-10. ?The fourth section is the preparation of bacterial genome DNAI. materialBacterial culture.Two,
27、equipmentPipette, high speed refrigerated centrifuge, tabletop centrifuge, water bath pot.Three reagents1, CTAB/NaCl solution: 4.1G, NaCl dissolved in 80ml, H2, O, slowly add 10g CTAB, add water to 100ml.2, other reagents: chloroform: isoamyl alcohol (24:1), phenol: chloroform: isoamyl alcohol (25:2
28、4:1), isopropanol, 70% ethanol, TE, 10% SDS,Protease K (20mg/ml or powder), 5mol/L, NaCl.Four, operation steps:1. 100ml bacteria overnight culture medium, 5000rpm centrifuged for 10 minutes, to supernatant.2., add 9.5ml, TE, suspension precipitation, and add 0.5ml 10% SDS, 50 L 20mg/ml (or 1mg dry p
29、owder) protease K, mix evenly, 37 hour heat preservation 1 hours.3. plus 1.5ml, 5mol/L, NaCl, mix evenly.4. add 1.5ml CTAB/NaCl solution, mix well, keep warm for 20 minutes at 65.5. with equal volume of phenol: chloroform: (25:24:1) extraction, 5000rpm centrifugation for 10 minutes, the supernatant
30、moved to clean centrifuge tube.6. extract with equal volume chloroform: isoamyl alcohol (24:1) and remove supernatant into the clean tube.7. and 1 times the volume of isopropanol, upside down mixing, 10 minutes at room temperature, precipitation DNA.8. remove the DNA precipitate with a glass rod, an
31、d then rinse after 70% ethanol rinse and dissolve at 1ml, TE and -20 DEG C for preservation. Such as DNA precipitation can not fish out, can be 5000rpm centrifugal, so that DNA precipitation.9. if you want to remove the RNA, you can follow the steps in section third of this chapter.Detection of fift
32、h genomic DNAThe DNA obtained by the above methods can generally be used for analysis of Southern, RFLP, PCR and so on. Because of the different materials used, the yield and quality of DNA are different. Sometimes, DNA contains phenols and polysaccharides, which will affect the effect of enzyme dig
33、estion and PCR. Therefore, the yield and quality of DNA must be detected after genomic DNA acquisition.After 1. DNA solution diluted 20-30 times, the ratio of OD260 and /OD280 was determined, and the content and quality of DNA were determined.2. l 2-5 agarose gel electrophoresis on 0.7% DNA gel to d
34、etect the size of the molecule.3. take 2 g DNA, cut with 10 units (U) Hind III enzyme overnight, 0.7% agarose gel electrophoresis, to detect whether the complete enzymatic hydrolysis (do RFLP, DNA must be fully enzymatic hydrolysis).If DNA contains many impurities, it can not be completely digested,
35、 or small molecules DNA, which affect the continuation of analysis and operation, can be dealt with in the following ways:(1) the selection of young plant tissues can reduce the starch content.(2) phenol: chloroform extract, remove protein and polysaccharide.(3) Sepharose column filtration was used to remove phenols, polysaccharides and small molecules DNA.(4) CsCl gradient centrifugation removes impurities and isolates large fragments of DNA (available for library construction).*JimiSoft:, Unregistered, Software, Convert, Part, Of, File, Read, Help, To, Know, ONLY, How, To, Register.*