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Preparation of Genomic DNA from E. coli

MOLECULAR BIOLOGY PROTOCOLS     

  • M.1:     Preparation of genomic DNA from bacteria
  • M.2:     PCR amplification of DNA
  • M.3:     Restriction enzyme digestion of DNA
  • M.4:     Phenol/chloroform extraction of DNA
  • M.5:     Ethanol precipitation of DNA
  • M.6:     Agarose gel electrophoresis
  • M.7:     Transformation of E. coli by electroporation
  • M.8:     Wizard PCR preps DNA purification system
  • M.9:     Alternate method for purifying DNA from agarose gels
  • M.10:   Transfection of mammalian cells using Lipofectamine (LTI)
  • M.11:   Southern blotting
  • M.12:   RT-PCR Protocol
  • M.13:   Preparation of sequencing gels
  • M.14:    Isolation of RNA using RNAzolTM
    •



    M.1: PREPARATION OF GENOMIC DNA FROM BACTERIA

    (Modified from Experimental Techniques in Bacterial Genetics, Jones and Bartlet, 1990)

    Materials:
     

    1.         Transfer 1.5 ml to a micro centrifuge tube and spin 2 min.  Decant the supernatant.  Drain well onto a Kimwipe.
    2.         Resuspend the pellet in 467 µl TE buffer by repeated pipetting.  Add 30 µl of 10% SDS and 3 µl  of 20 mg/ml
                proteinase K, mix , and incubate 1 hr at 37 C.
    3.         Add an equal volume of phenol/chloroform and mix well by inverting the tube until the phases are completely
                mixed.    CAUTION:  PHENOL CAUSES SEVERE BURNS, WEAR GLOVES GOGGLES, AND LAB COAT
                AND KEEP TUBES CAPPED TIGHTLY.  Carefully transfer the DNA/phenol mixture into a Phase Lock GelTM tube
                (green) and spin 2 min.
    4.         Transfer the upper aqueous phase to a new tube and add an equal volume of phenol/chloroform.  Again mix well and
                transfer to a new Phase Lock GelTM tube and spin 2 min.  Transfer the upper aqueous phase to a new tube.
    5.         Add 1/10 volume of sodium acetate.
    6.         Add 0.6 volumes of isopropanol and mix gently until the DNA precipitates.
    7.         Spool DNA onto a glass rod (or Pasteur pipet with a heat-sealed end).
    8.         Wash DNA by dipping end of rod into 1 ml of 70% ethanol for 30 sec.
    9.         Resuspend DNA in 100-200 µl TE buffer.
    10.       After DNA has dissolved, measure the concentration by diluting 10 µl of DNA into 1 ml of TE (1:100 dilution) and
                measure absorbance at 260 nm.  Concentration of original DNA solution in µg/ml = Abs x 100 x 50 µg/ml.