基因组DNA的提取与纯化

Extraction and purification of genomic DNA in Nai(Prunus salicina Lindl.var.cordata)

以嫩芽为材料,对Dellaporta等用CTAB制备植物基因组DNA的方法进行改良,结果表明,加入质量分数ω=10%PVPP与材料充分研磨,将提取缓冲液的β-巯基乙醇体积分数降低为1%,能有效地去除材料中的多酚类物质;用氯仿/异戊醇(24∶1)抽提裂解液2次所获得的上相,加入1/10体积的65℃的NaCl/CTAB溶液混匀,再用氯仿/异戊醇(24∶1)连续抽提2次,并在DNA粗提液中加入适量高浓度NaCl和无水乙醇沉淀DNA,可以有效地去除多糖的干扰,获得比较纯净的DNA样品;PCR特异性扩增的结果表明,以改良法的DNA为模板可获得PPO基因保守区约600～800bp特异条带,且扩增条带较亮、无引物二聚体出现。

Nai's genomomic DNA from young buds was extracted by modified Dellaporta'method for preparing genomic DNA of plants with Cetyl Triethyl Ammonium Bromide (CTAB). The results showed that polyphenolics were effectively removed from materials by blendering 10% Poly Vinyl Poly Pyrrilodone (PVPP) to the ground materials under liquid nitrogen in mortar and lowering concentration of β-mercaptoethanol in extracting buffer to 1%; the polysaccharides were effectively removed and purer DNA samples were obtained by blundering aqueous phase after extracting lysis solution twice with 24∶1 chloroform:isoamyl alcohol with 1/10 volume NaCl/CTAB solution preheated by 65℃ then successively extracting twice with 24∶1 chloroform:isoamyl alcohol and adding an appropriate amount 5 mol/L sodium chloride and absolute ethanol to the rough solution of DNA.PCR amplication was conducted by using obtained DNA samples as templates, a blighter 600～800bp special band of conserve zone in PPO gene was amplified and no primer dimers emerged.