Characteristics of this procedure: |
PROCEDURE The protocol is slightly modified from the procedure described in the original paper (only with respect to agarose gel concentration, voltage and running time).Harvest cells by trypsinization (about 2x106 cells). Spin cells down at 200xg for 5 min. Resuspend cells in 1 ml Hanks' buffered salt solution (HBSS) Transfer the cells into 10 ml of ice-cold 70% ethanol; store the cells at -20°C for 24 h or longer Spin down fixed cells at 800 g for 5 min and remove the ethanol thoroughly! Resuspend the cell pellet in 40 µl phosphate-citrate buffer (PCB), consisting of 192 parts of 0.2 M Na2HPO4 and 8 parts of 0.1 M citric acid (pH 7.8). Incubate at RT for at least 30 min. Spin cells down at 1000 g for 5 min. Transfer supernatant to new tubes. Optionally, the samples can be concentrated by vacuum in a SpeedVac concentrator. Add 3 µl of 0.25% Nonide NP-40 (in water) and 3 µl of RNase (1 mg/ml in water) to the samples and incubate for 30 min at 37°C. Add 3 µl of proteinase K (1 mg/ml) to the samples and incubate for another 30 min at 37°C. Add 12 µl of loading buffer (0.25% bromophenol blue, 30% glycerol) to the samples and load on a 1.5% agarose gel. Run the gel at 4 V/cm for about 4 hours and detect DNA by ethidium bromide under UV light. |