5.1.1. Large-Scale Genomic DNA Extraction Protocol

NOTE: Use of disposable tubes is highly recommended in order to avoid contamination.

1. 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).

2. Add 0.25 ml 10% SDS, mix, and incubate 5 minutes at room temperature.

3. 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.

4. Add 10 μl of Proteinase K, mix, and incubate 15 minutes at room temperature.

5. 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).

6. 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).

7. 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.

8. 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.

9. Discard supernatant and air-dry pellet.

10. Dissolve DNA pellet in an appropriate volume of dH₂O to a concentration of ~2 mg/ml.

11. Incubate 30 minutes at 80°C before spectrophotometer reading.

NOTE: Expected yield is about 10 μg genomic DNA per 1 million cells.