Isolating Phage Genomic DNA

This protocol has been tested with phage T7.

Materials

• 5× Phage precipitation buffer (20% w/v PEG 8000, 2.5 M NaCl)
• Phage resuspension buffer (1M NaCl, 10mM Tris pH 7.5, 0.1 mM EDTA)
• DNase I (20 mg/mL)
• RNase A (20 mg/mL)

Procedure

1. Transfer 4-8 mL of phage lysate (do not include any chloroform) to a new 15 ml tube. Add 1/5 the volume of 5× phage precipitation solution (20% w/v PEG 8000, 2.5 M NaCl) and mix well by inverting the tube.
2. Incubate this mixture for at least 2 h at 4°C
3. Centrifuge for 30 min at 10,000×g at 4°C
4. Pour off the supernatant and add
5. Add 1 mL of T7 phage resuspension buffer followed by 1 µL DNase I (20 mg/mL) and 1 µL RNase A (20 mg/mL). Incubate for 30 minutes at 37°C to degrade any remaining bacterial nucleic acids.
6. (Add EDTA to soak up whatever is in the buffer)
7. Add 50 µg/mL Proteinase K and incubate at 65°C.
8. Phenol-chloroform extract
9. Ethanol precipitate
10. Resuspended in 10 mM Tris HCl pH 8.5 for analysis