Preparation of DNA From Cells in 24-WELL Tissue Culture Plates

Preparation of DNA From Cells in 24-WELL Tissue Culture Plates

*The Qiagen DNeasy kit works well for prepping DNA from cells.

The following protocol is taken directly from Manipulating the Mouse Embryo, 3rd Edition, Nagy et al.

Materials:

• Cells: Confluent ES cell cultures in gelatinized 24 or 4-well tissue culture plates
• Equipment:
  • Microfuge tubes, 1.5ml
  • Micropipettor, 200-1000ul
  • 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), and sodium acetate (3M, pH 5.2)

Procedure:

1. Aspirate the medium from each plate containing confluent ES cells, rinse the wells once with cold PBS, add 200-500ul of lysis buffer per well, 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 volumes of phenol/chloroform/ iso-amyl alcohol to the tube and 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 50-100ul of TE buffer, and incubate for 3-4 hours at 55°C to dissolve. Store at 4°C.