马尾松针叶DNA提取方法研究

DNA Isolation from Pinus massoniana Needles

采用3种不同的方法(CTAB法、SDS法和试剂盒法)提取马尾松幼嫩针叶基因组DNA,并分别从取样材料类型、样品的保存、提取液中PVP含量、取样质量、抽提过程中提取程序的改进等方面进行提取马尾松基因组DNA效果的凝胶电泳比较.结果表明:CTAB法适合于在马尾松群体分子生物学研究中基因组DNA的提取:采用低温冷藏法保存的春季抽梢针叶0.2 g,在CTAB的提取液中加入2%的PVP,常规CTAB法提取程序中添加等体积的混合液(V酚∶V氯仿∶V异戊醇=25∶24∶1)并在此前加入1/10体积的10×CTAB,可以有效地提高马尾松基因组DNA的提取质量,获得显带平直、纯度较高的马尾松基因组DNA,OD260/OD280值在1.8左右.这为马尾松群体分子生物学的研究提供了一个经济、简便而有效的DNA提取方法.

Three methods （CTAB, SDS and plant DNA kit） were used to extract genomic DNA from the needles of Pinus massoniana. The results show that the modified CTAB was suitable for this extraction. Its quality was influenced by several factors ： materials type and quantity, storage, the content of PVP in the extraction buffer and the initial mass amount, and the modification of isolating procedures. And the results also indicate that the quality can be improved during the CTAB DNA extraction if emphasis be placed on the following steps： 1） Using 0. 2 g young needles in spring; 2） Storing the needles in low-temperature; 3） employing 2% PVP in the extraction buffer; 4） Adding two processes of 1/10 of 10 × CTAB and the same volume of the extraction buffer with phenol / chloroform / isoamyl alcohol （25 ; 24 ; 1 ,v/v） before the DNA sedimentation, which can effectively remove such impurities as poly-phenols, lipids and polysaccharides and then get a straight and bright DNA band on the 0. 8% gelose electrophoresis. This provides an economic, convenient and effective method of DNA isolation for a biological study of Pinus massoniana.