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本文介绍了福建柏Fokieniahodginsii(Dunn)HenryetThomas总DNA的提取介质组成分 ,建立了一个快速、简便且高效提取和鉴定福建柏鲜叶、干叶、种子总DNA的方法。该方法所提DNA的产率分别为 :1 5 0～ 2 5 0 μg/g鲜叶 ,1 0 0～ 1 5 0 μg/g干叶和 1 4 0～ 1 6 0 μg/g种子 ;绝大多数粗提总DNAOD2 6 0 /OD2 80 =1 .80±0 .0 2 ;提取的鲜叶、种子和单种子胚乳总DNA皆为 4 8kb,而干叶总DNA为 5 0Kb ,都适于限制性酶切和RAPD反应 ;一般实验时间为 4h。该方法既不需氯化铯梯度离心和柱层析纯化 ,也不需氯仿 :异戊醇抽提 ,粗提的总DNA样品不经RNase消化即可用于RAPD反应或经RNase消化用于限制性酶切 ,因此 ,具有高产率、高纯度、高质量和快速简便等优点。尤其是它所提取的干叶DNA ,适于限制性酶切和PCR反应 ,解决了取材上的不便。实验中还发现 :( 1 )提取DNA之前去除叶绿素与否 ,对提取福建柏总DNA无明显影响。 ( 2 )只有去除RNA的福建柏总DNA样品 ,才能被限制性内切酶完全切开。 ( 3)RAPD模板中含RNA及一定量的蛋白质 ,均不影响扩增效果。 ( 4 )同一个体的鲜叶、干叶、种子总DNA样品 ,皆可得到完全一致的扩增产物。
In this paper, the components of total DNA extracted from Fokieniahodginsii (Dunn) HenryetThomas were introduced, and a rapid, simple and efficient method for the extraction and identification of the total DNA of fresh leaves, dry leaves and seeds of Fokienia hodginsii (Dunn) was established. The yield of DNA extracted by this method was: 150 ~ 250 μg / g fresh leaves, 100 ~ 150 μg / g dry leaves and 140 ~ 160 μg / g seeds, respectively Most of the total crude DNAOD2 60 / OD2 80 = 1.80 ± 0. 0 2; extracted fresh leaves, seeds and single seed endosperm total DNA were 48kb, and dry leaf total DNA of 50Kb, are suitable Restriction enzyme digestion and RAPD reaction; the general experiment time is 4h. This method neither cesium chloride gradient centrifugation and column chromatography purification, nor chloroform: isoamyl alcohol extraction, the crude total DNA sample without RNase digestion can be used for RAPD reaction or RNase digestion for restrictive Digestion, therefore, has the advantages of high yield, high purity, high quality and quick and easy. In particular, it is extracted from the dry leaf DNA, suitable for restriction and PCR reaction, to solve the inconvenience of drawing. The experiment also found: (1) removal of chlorophyll before or after DNA extraction had no significant effect on the total DNA extracted from Fujian cypress. (2) Only the total DNA samples of Fujian cytoplasm with RNA removed can be completely excised by restriction enzyme. (3) RAPD template containing RNA and a certain amount of protein, do not affect the amplification effect. (4) the same individual fresh leaves, dry leaves, total DNA samples of seeds, can be exactly the same amplification products.