Can any one clarify my question with DNA

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    • #9280
      shivakumar
      Participant

      hi everybody

      while extracting DNA, extraction buffer containing
      NaCl, EDTA, Tris n CTAB
      whts d role of those chemicals n
      n why adjusting d buffer pH to 8

      n I feel same doubt with chloroform isoamyl alcohol(their role)

    • #82830
      canalon
      Participant

      I can clarify your question. I would use good syntax and complete words. It would like that:

      "In a protocol for the extraction of DNA we use the following reagents, could you tell me what are their different roles? NaCl, EDTA, Tris, CTAB, Chloroform/Isoamyl alcohol.
      Similarly why is the pH adjusted to 8?"

      Now as a beginning of an answer:
      NaCl is used to prevent the co-precipitation of DNA and CTAB.
      EDTA is used to chelate divalent cations, which are necessary (among other things) for proper DNASe activity
      Tris is a Buffer
      CTAB is a cationic detergent that precicpitates polysaccharides
      Chloroform is usually used with phenol to denature proteins. IAA is just here to improve the separation of the aqueous and polar phases
      DNA degrades in acidic environment (depurination)

    • #82832
      Cat
      Participant

      pH 8 as far as I know is optimal for the extraction and was determined experimentally (correct me if I am wrong).

      NaCl and CTAB – used together to remove polysaccharides
      EDTA – chelating agent, used to remove Mg2+ and Ca2+ from DNase enzymes and prevent DNA degradation.
      TRIS – keeps DNA soluble in water by keeping it deprotonated
      Phenol:chloroform isoamyl alcohol – to remove proteins

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