用改进的高盐低pH法分离和纯化棉花叶绿体及叶绿体DNA

Purification of Cotton Chloroplast and Chloroplast DNA Using the Improved Method of High Salt Concentration and Low pH

报道了用改进的高盐低ｐＨ法分离和纯化棉花叶绿体及叶绿体ＤＮＡ的实验过程，即先在高盐（离子强度）介质下，通过ｐＨ值变换，多次洗涤的方法去除叶绿体表面的静电附着杂质，得到纯净的叶绿体；然后氯仿抽提纯化ＤＮＡ；最后采用界面针挑法得到高纯度的ｃｐＤＮＡ。实验过程中针对棉花的特点，采用ＰＶＰ排除棉酚和丹宁的干扰，用ＤＩＥＣＡ抑制酚氧化酶活性，并对实验细节作了较大改进。得到的ＤＮＡ可满足酶切、ＰＣＲ等分子生物学分析。

The improved method of high salt concentration and low pH suitable for extraction of cotton chloroplast and chloroplast DNA(cpDNA) is described in details in this protocol. The chloroplasts are purified by washing for several times in high salt concentration (ionic strength) and varied pH medium. The proteins are deleted by extraction with an equal volume of chloroform. At last, the highly purified cpDNA is picked out from the interphase of the ethanol and the supernatant using a hooked needle. PVP and DIECA are used to bind phenolic compounds and tanin and to inactivate phenol oxidases respectively. We have successfully used cpDNA prepared by this method for restriction analysis, PCR and other molecular operations.