Isolation of high molecular weight DNA from culture cells

  • 1st day:
    • centrifuge a sufficient volume of cell culture (several 20 ml) for 10 min at 1000 rpm at room temperature
    • discard the supernatant and resolve the pellet in 500 µl sterile TBS (100 mM NaCl2, 20 mM TRIS/Cl, 10 mM EDTA, pH 7.5) or PBS
      • remove 10 µl and add to this aliquote 90 µl of trypan-blue solution (10fold dilution)
      • count the colorless (living) cells of 1 µl (5 great squares of a Malasset counting chamber x 20)
      • multiply by 5000 to obtain the total number of cells in the tube
      • divide this number by 25 x 106 to get the volume in ml in which finally the cells are resuspended
    • wash the pellet 2 times in 10 ml sterile TBS at room temperature
    • resuspend the pellet in the above calculated volume of TBS
    • prepare 1% low melting agarose in TBS
    • prepare clean 80 µl slots on ice
    • mix equal volumes of liquid agarose and cell suspension
    • immediately pour the mixture into the slots and let the agarose solidify
    • transfer the agarose blocks into the lysis solution (0.5 M EDTA, 1 % SDS, 2 g/l Protease K)
    • incubate for 48 hours at 50°C
  • 3rd day:
    • wash the agarose blocks three times in approximately 10 fold their volume sterile TE
    • incubate each time at 50°C for 30 min
    • transfer into TE and leave at 4°C
  • 4th day:
    • replace the TE and leave at 4°C
  • 5th day:
    • replace the TE and leave at 4°C
  • 6th day:
    • if the agarose block are translucent replace the TE by 0.5 M EDTA pH 8 and store the DNA at 4°C

    Solutions:

    TBS

    100 mM NaCl2, 20 mM TRIS/Cl, 10 mM EDTA, pH 7.5

    lysis solution

    0.5 mM EDTA pH 9s, 1 % SDS, 2 g/l Protease K

    TE

    10 mM TRIS, 0.5 mM EDTA, pH 7.5

    storage solution

    0.5 M EDTA , pH 8

    trypan-blue solution

    3 volumes of 0.1 % trypan-blue stock + 1 volume 25.5 g/l NaCl in TBS (hypertonic TBS)