SDS法提取石斛基因组DNA的研究

以密花石斛（Dendrobium densiflorum Lindley ex Wallich）为研究试材,对植物SDS方法提取基因组DNA中的若干影响因子诸如药品、试剂配制方法、DNA取材部位、蛋白质去除次数、DNA析出时间、提取样品水浴时间等进行了比较分析。结果表明：不同的SDS缓冲液配制方法提取效果差异较大,Wang＇s配方产率及纯度都较高;石斛花苞的DNA提取产率较叶片高,但纯度较叶片低;提取过程中,适宜的水浴时间当为30～40min,过短DNA产率下降,过长会引起DNA的降解,或最后产物中增加其他杂质;氯仿/异戊醇抽提次当为2～3次,对去除DNA中蛋白质、多糖及其它杂质有一定的作用,过多则DNA损失也明显;异丙醇沉淀时间10min时核酸析出量在80%以上,纯度相对较高,继续沉淀的产物中DNA含量及纯度明显变低,同时采取挑取絮状沉淀或再用无水乙醇纯化10min的方法,可减少蛋白质、多糖等其它杂质的影响。

Extraction of Genomic DNA of Dendrobium with SDS

Genomic DNA of Dendrobium densiflorum Lindley ex Wallich was extracted with SDS, and some influential factors affecting extraction of plant genomic DNA, such as chemicals, reagent preparation method, part of material for DNA extraction, deproteinizing frequency, time for DNA precipitation, water bathing time for the sample extracts, and so on were compared and analyzed to find an optimum method for extracting of genomic DNA of D. densiflorum. SDS buffers had much different effects on DNA extraction, and Wang＇s SDS buffer gave higher DNA yield and purity. The flower buds of D. densiflorurn had a higher DNA yield than the leaves but lower DNA purity. Samples placed in water bath for 30-40 min yielded higher in DNA. DNA yield was lower when the samples were placed in water bath for too short a time, and DNA degeneration occurred or DNA products had more other impurities when the samples were placed in water bath for too long a time. DNA was extracted 2-3 times with chloroform/ isoamyl alcohol, which was conducive to removing of the proteins, the polysaccharide and other impurities from the DNA. Genomic DNA loss occurred obviously when extraction frequency was high. Precipitation with isopropyl alcohol for 10 min yielded more than 80% of genomic DNA with higher purity, but the ge nomic DNA content and purity in the precipitate were evidently reduced when the precipitation continued.Meanwhile the flocculent DNA precipitate was collected or purified for 10 min with absolute ethanol to reduce protein, polysaccharide and other impurities.