提取DNA为什么需在碱性条件下

2025-04-03 11:01:20
推荐回答(1个)
回答1:

提取DNA需要在碱性条件下的原因如下:

在酸性条件下,DNA容易被水解,但最不稳定是的嘌呤与脱氧核糖之间的糖苷键.在碱性条件下,RNA由于2'-OH的存在,比DNA要容易水解得多,DNA在碱性条件下相对稳定.


DNA提取方法:

  1. 酚抽提法:先用蛋酶K、SDS破碎细胞,消化蛋白,然后用酚和酚-氯DNA大小为100-150kb

  2. 甲酰胺解聚法:破碎细胞同上,然后用高浓度甲酰胺解聚蛋白质与DNA的结合,再透析获得DNA可得DNA200kb左右。

  3. 玻璃棒缠绕法:用盐酸胍裂解细胞,将裂解物铺于乙醇上,然后用带钩或U型玻璃棒在界面轻搅,DNA沉淀液绕于玻棒。生成DNA约80kb。

  4. 异丙醇沉淀法:基本同1法,仅用二倍容积异丙醇替代乙醇,可去除小分子RNA(在异丙醇中可溶状态)

  5. 表面活性剂快速制备法:用Triton X-100A或NP40表面活性剂破碎细胞,然后用蛋白酶K或酚去除蛋白,乙醇沉淀或透析。

  6. 加热法快速制备:加热96℃-100℃,五分钟,然后离心后取上清,可用于PCR反应。

  7. 碱变性快速制备:先用NaOH作用20分钟,再加HCI中和,离心后取上清,含少量DNA。

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