Preparation of DNA from Cells in 60mm Tissue Culture Dishes
- Cells: Confluent ES cell cultures in gelatinized 60mm tissue culture dish
- Microfuge tubes, 1.5ml
- Micropipettor, 200-100ul
- Rocking platform or orbital shaker
- Ethanol, 70%
- Lysis buffer:
100mM Tris-HCl (pH8.5)
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)
- 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.
- 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).
- Remove the DNA using the disposable tip of a micropipettor or a glass rod and place the DNA into Eppendorf tubes. Spin briefly to pellet.
- 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.