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2 ml离心管法快速制备水稻高质量总DNA   

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实验原理:CTAB (hexadecyltrimethylammonium bromide,十六烷基三甲基溴化铵),是一种阳离子去污剂,它与DNA结合形成的复合物在高盐溶液中可溶并且稳定存在。在粉碎后的植物叶片组织中加入含有CTAB的抽提缓冲液,可以保持DNA溶解于水相。用氯仿除去蛋白质,多糖和酚类等有机杂质后,在水相中加入异丙醇即可将纯度较高的DNA沉淀出来。
实验目的:快速制备少量可用于酶切连接反应的水稻总DNA。

关键词: 水稻, 叶片, 抽提, 高质量DNA

材料与试剂

  1. CTAB (Bio Basic Inc., Canada)
  2. NaCl (国药试剂)
  3. EDTA (天津市天力化学试剂有限公司)
  4. Tris (Angus Chemical Company, USA)
  5. HCl (国药试剂)
  6. RNase A (Invitrogen, USA)
  7. β-巯基乙醇 (Sigma, USA)
  8. 三氯甲烷 (国药试剂)
  9. 异丙醇 (国药试剂)
  10. 液氮 (武汉液氮厂)
  11. 95%乙醇 (国药试剂)
  12. CTAB抽提缓冲液 (见溶液配方)
  13. TE缓冲液 (见溶液配方)

仪器设备

  1. 台式离心机 (Beckman, USA)
  2. 电热恒温培养箱 (SKP-02.3000,黄石市恒丰医疗器械有限公司)
  3. 冰箱 (BCD-243K F&R,新飞)

实验步骤

  1. 在苗期收获适量水稻叶片于-20 °C冰箱保存备用。
  2. 将叶片在液氮中磨碎,转移1/4到1/3管量的粉末到2 ml离心管中。
  3. 向每管样品中加入800 μl CTAB抽提缓冲液,振荡1 min,65 °C温育10-15 min。
  4. 加0.7倍体积三氯甲烷(600 μl),剧烈振荡1 min。
  5. 12,000 rpm离心10 min,将上清液(500 μl)转移的新的1.5 ml离心管中。
  6. 加0.7倍体积异丙醇(350 μl),颠倒混匀,12,000 rpm离心10 min。
  7. 倒上清液,加70%乙醇1 ml洗沉淀。
  8. 倒乙醇,在超净台上将沉淀吹干,加50 μl TE溶解。
  9. 用1%(w/v)琼脂糖凝胶电泳检测DNA质量(选做)。

注意事项

  1. 用本法抽提的DNA质量较高,但有部分降解是正常现象 (图1)。一般主带处未降解的完整DNA浓度约为140-200 ng/μl (与上样量有关)。


    图1. 2 ml离心管法抽提的DNA电泳检测图

  2. 在-70 °C超低温冰箱保存样品,磨样前叶片不冻融可以显著提高DNA质量。
  3. 叶片粉末磨得越细,最后抽提到的DNA质量越好,产量越高。

溶液配方

  1. CTAB抽提缓冲液
    2% (w/v) CTAB
    1.42 mol/L NaCl
    20 mmol/L EDTA
    100 mmol/L Tris, pH 8.0
    使用前加1/80体积RNase A, 0.2% (v/v) β-巯基乙醇
  2. TE缓冲液
    10 mmol/L Tris, pH 8.0
    1 mmol/L EDTA
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:龙湍, 杨莹, 符德保, 吴昌银. (2018). 2 ml离心管法快速制备水稻高质量总DNA. Bio-101: e1010106. DOI: 10.21769/BioProtoc.1010106.
How to cite: Long, T., Yang, Y., Fu, D. B. and Wu, C. Y. (2018). A Rapid Method for Isolation of High-quality Total DNA Using 2 ml Centrifugal Tube. Bio-101: e1010106. DOI: 10.21769/BioProtoc.1010106.
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