細菌基因組DNA的提取方法綜述

1 快速微量提取法
      A.取1.5ml菌體培養物于一滅菌Ep管中，12000rpm離心1min, 丟去上清夜，收集菌體。
      B.加入400ul裂解液（40mMTris-醋酸，20mM醋酸鈉,1mMEDTA,1%SDS,pH7.8）混勻,置于37oC水浴1hr。
      C.然后加入200ul5mol/L的氯化鈉溶液，混勻后于13000rpm離心15min。
      D.取上清液，用苯酚抽提2次，氯仿抽提1次。
      E.加兩倍體積無(wú)水乙醇，1/10體積醋酸鉀(3M ,pH8.0)，-20度保存1小時(shí)后，13000rpm離心15min，棄上清液，沉淀用70%乙醇洗2次；置于室溫干燥后，溶于50ulTE溶液中，置4oC保存備用。

2 蛋白酶/SDS法制備
      先用10ml含適當抗生素的GBM過(guò)夜培養Delftia sp.，第二天4000rpm離心10min收集菌體，用Washing TE（50mmol/LTris-HCl pH8.0,10mmol/LEDTA pH8.0）洗菌體2次，之后將菌體充分懸浮在5ml 1×TE緩沖液中，先后加入0.5ml 5mg/L的蛋白酶、0.5ml 10% SDS，輕輕混勻后50℃放置3h~5h，接著(zhù)用等體積的Tris飽和苯酚抽提2次，苯酚/氯仿/異戊醇抽提一次，氯仿抽提一次后，乙醇沉淀DNA，用自動(dòng)移液器吸管頭將絮狀DNA沉淀塊吸附到Ep管中，70%乙醇洗2次，干燥后溶于適當1×TE或ddH2O中。

3 DNA EXTRACTION PROCEDURE - GENERAL
      1) Grow cells overnight in 500 ml broth medium.
      2) Pellet cells by centrifugation, and resuspend in 5 ml 50 mM Tris (pH 8.0), 50 mM EDTA.
      3) Freeze cell suspension at -20C
      4) Add 0.5 ml 250 mM Tris (pH 8.0), 10 mg/ml lysozyme to frozen suspension, and let thaw at room temperature. When thawed, place on ice for 45 min.
      5) Add 1 ml 0.5% SDS, 50 mM Tris (pH 7.5), 0.4 M EDTA, 1 mg/ml proteinase K. Place in 50C water bath for 60 min.
      6) Extract with 6 ml Tris-equilibrated phenol and centrifuge at 10,000X g for 15 min. Transfer top layer to new tube (avoid interface). Re-do this step if necessary.
      7) Add 0.1 vol 3M Na acetate (mix gently), then add 2 vol 95% ethanol (mix by inverting).Spool out DNA and transfer to 5 ml 50 mM Tris (pH 7.5), 1 mM EDTA, 200 g/ml RNase. Dissolve overnight by rocking at 4C.
      8) Extract with equal volume chloroform (mix by inverting) and centrifuge at 0,000X g for 5 min. Transfer top layer to a new tube.
      9) Add 0.1 vol 3M Na acetate (mix gently), then add 2 vol 95% ethanol (mix by inverting).
      10) Spool out DNA and dissolve in 2 ml 50 mM Tris (pH 7.5), 1 mM EDTA.
      11) Check purity of DNA by electrophoresis and spectrophotometric analysis.

  （本文轉載丁香園）