植物葉片經(jīng)液氮研磨,可使細(xì)胞壁破裂,加入去污劑（如CTAB）,可使核蛋白體解析,然后使蛋白和多糖雜質(zhì)沉淀,DNA進(jìn)入水相,再用酚、氯仿抽提純化.本實驗采用CTAB法,其主要作用是破膜.CTAB是一種非離子去污劑,能溶解膜蛋白與脂肪,也可解聚核蛋白.植物材料在CTAB的處理下, 結(jié)合 65℃ 水浴使細(xì)胞裂解、蛋白質(zhì)變性、DNA 被釋放出來.CTAB與核酸形成復(fù)合物,此復(fù)合物在高鹽（>0.7mM）濃度下可溶,并穩(wěn)定存在,但在低鹽濃度（0.1-0.5mM NaCl）下CTAB-核酸復(fù)合物就因溶解度降低而沉淀,而大部分的蛋白質(zhì)及多糖等仍溶解于溶液中.經(jīng)過氯仿 / 異戊醇 (24:1) 抽提去除蛋白質(zhì)、多糖、色素等來純化DNA,最后經(jīng)異丙醇或乙醇等沉淀劑將DNA沉淀分離出來.
Plant leaves by grinding in liquid nitrogen, can make the cell wall broken, adding detergent (such as CTAB), can make nuclear protein analysis, and then theprotein and polysaccharide impurity precipitation, DNA into the aqueous phase,and phenol chloroform extraction, purification. This experiment adopts the CTABmethod, its main role is to rupture of membranes. CTAB is a nonionic detergent,can dissolve the membrane protein and fat, can also be depolymerized nuclearprotein. Plant materials in CTAB treatment, combined with 65 ℃ water bath to cell lysis, protein denaturation, DNA is released. CTAB and nucleic acid complexes, thecomplexes in high salt (>0.7mM) concentration and soluble, stable, but at low salt concentration (0.1-0.5mM NaCl) CTAB- nucleic acid complexes because ofreduced solubility and precipitation, and most of the protein and polysaccharide isdissolved in solution. By chloroform / isoamyl alcohol (24:1) extraction to remove proteins, polysaccharides, pigments for purification of DNA, and finally by isopropyl alcohol or ethanol as precipitant DNA precipitation separated.
由于核酸、蛋白質(zhì)、多糖在特定的紫外波長都有特征吸收.核酸及其衍生物的紫外吸收高峰在260nm.純的DNA樣品A260/280≈1.8,純的RNA樣品A260/280≈2.0,并且1μg/ml DNA溶液A260=0.020.
Because the nucleic acid, protein, polysaccharide has the characteristic absorptionin the UV wavelength specific. Nucleic acid and its derivative UV absorption peak at 260nm. Pure DNA samples with A260/280 ≈ 1.8, pure RNA sample A260/280 ≈ 2,and 1 μ g/ml DNA solution A260=0.020