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Houston, Texas

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Mouse Embryonic Stem Cell Core Facility
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Preparation of DNA from Cells in 60mm Tissue Culture Dishes

Materials:

  • Cells: Confluent ES cell cultures in gelatinized 60mm tissue culture dish
  • Equipment:
    • Microfuge tubes, 1.5ml
    • Micropipettor, 200-100ul
    • Rocking platform or orbital shaker
  • Reagents:
    • Ethanol, 70%
    • Isopropanol
    • Lysis buffer:
      100mM Tris-HCl (pH8.5)
      5mM EDTA
      0.2% SDS
      200mM NaCl
      100ug/ml Proteinase K (20mg/ml Proteinase K stock is stored at -20°C and added to the lysis buffer prior to use)
    • Phosphate-buffered Saline (PBS)
      TE Buffer (10mM Tris-HCl [pH 8.0], 1mMEDTA)

      Optional: 100% Ethanol (kept at -20°C Phenol:Chloroform:Iso-amyl alcohol (25:24:1)

Procedure:

  1. Aspirate the medium from each plate containing confluent ES cells, rinse the plates once with 4mls PBS, add 500ul lysis buffer per plate, and incubate for 4-16 hours in humid conditions at 55°C. Optional: Harvest cells by trypsinization, transfer cell suspension to microfuge tubes, wash with PBS, add 500ul lysis buffer per sample, and incubate for 4-16 hours at 55°C. It is also possible to collect cells by adding the lysis buffer directly to the cells. After the incubation, add equal volume of phenol/chloroform/ iso-amyl alcohol to the tube vortex for 1 minute. Centrifuge at 14,000 rpm for 5 minutes. Carefully transfer the top layer (water phase) to a new tube and add 1/10 volume of 3M sodium acetate and 3 volumes of 100% ethanol (-20°C). Centrifuge at 14,000 rpm for 10 minutes, remove supernatant, and continue with step 4.
  2. After the incubation, shake the plate on a shaker for 15 minutes, add equal volume of isopropanol per plate and shake until DNA precipitate becomes visible (15-30 minutes).
  3. Remove the DNA using the disposable tip of a micropepettor or a glass rod and place the DNA into Eppendorf tubes. Spin briefly to pellet.
  4. Wash the DNA pellet two times with 70% ethanol and dry for 10 minutes at room temperature. Resuspend the DNA pellet in 300-500ul of TE buffer, and incubate for 3-4 hours at 55°C to dissolve. Store at 4°C.