NOTE: Use of disposable tubes is highly recommended in order to avoid contamination.
- Resuspend cell pellet in 5 ml QIAGEN Buffer P1 (with RNase A) in a 15 ml polypropylene (phenol:chloroform resistant), Falcon screw-cap centrifuge tube (12,000 RCF rated).
- Add 0.25 ml 10% SDS, mix, and incubate 5 minutes at room temperature.
- Using an ultrasonic homogenizer, sonicate to shear DNA into 10-100 kb sized fragments. To prevent cross-contamination, thoroughly wash the ultrasound head with running water and dry with clean paper towels between samples.
- Add 10 μl of Proteinase K, mix, and incubate 15 minutes at room temperature.
- Add 5 ml Phenol:Chloroform:Isoamyl Alcohol solution, vortex hard, and spin down 60 min, 20°C at 8,000 rpm in JA-14 or equivalent rotor (Beckman).
- At this point, there should be approximately 5 ml of clear upper phase. Transfer 4 ml of upper phase to a new 15 ml disposable screw cap tube (same as in Step 1).
- Add 0.5 ml of 3M Sodium Acetate and 4 ml isopropanol, mix well, then spin down 30 min, 20°C at 8,000 rpm in a JA-14 or equivalent rotor.
NOTE: To produce a more visible pellet that is compacted at the bottom of the tube, it is recommended to incubate overnight at room temperature before centrifugation.
- Discard supernatant, add 10 ml of 70% ethanol, and spin down 5 min, 20°C at 8,000 rpm in a JA-14 or equivalent rotor.
- Discard supernatant and air-dry pellet.
- Dissolve DNA pellet in an appropriate volume of dH2O to a concentration of ~2 mg/ml.
- Incubate 30 minutes at 80°C before spectrophotometer reading.
NOTE: Expected yield is about 10 μg genomic DNA per 1 million cells.
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