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    • #1109
      chemistry_freako
      Participant

      In the purification of plasmid DNA (of E.coli), 6 reagents were used:
      Cell resuspension solution, cell lysis solution, alkaline protease, neutralising solution, column wash solution, as well as nuclease free water.

      Components of the reagents are as such:
      Cell resuspension solution:
      Tris-HCl
      EDTA
      RNase A

      Cell lysis solution:
      NaOH
      SDS

      Alkaline protease
      Protease enzyme subtilisin (produced by Bacillus licheniformis)

      Neutralising solution:
      Potassium acetate
      Tris- HCl
      EDTA

      Column wash solution:
      potassium acetate
      Tris- HCl
      EDTA

      So what exactly is the function of all the various components, of the various reagents, in obtaining a purified sample of the plasmid DNA puc19 from E.coli?

    • #24106
      DevGrp
      Participant

      this site should help

      http://lsvl.la.asu.edu/resources/mamaji … aline.html

      Never used Protease myself, and not sure why you have a “Column wash solution” step

      And generally Tris is a pH buffer and EDTA celates metal ions which inhibits nucleases.

    • #24329
      chemistry_freako
      Participant

      i see…thanks!

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