Isolating Phage Genomic DNA

This protocol has been tested with phage T7. It has a double-stranded genome that has a length of 40 kb.

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)
• 0.5 M EDTA (pH 8.0)

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. This step is very important for degrading any remaining nucleic acids from lysed bacterial cells!
6. Add 0.5 M EDTA (pH 8.0) to chelate divalent metals in the buffer (how much?)
7. Phenol-chloroform extract: Add 1 volume of XXXXXXXX
8. Ethanol precipitate or run through a PCR cleanup column to exchange the buffer
9. Resuspend in 10 mM Tris HCl pH 8.5 for storage.
10. Measure the concentration of DNA in your sample. The T7 genome is double-stranded DNA.

Notes

• T7 DNA is large (40 kb). If you want your DNA to remain (mostly) intact, you need to be careful: avoid vortexing or pipette very gently after the phenol-chloroform extraction step.
• An alternative DNA purification method that may give higher quality DNA is performing centrifugation with a cesium chloride gradient.