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 solution (20% w/v PEG 8000, 2.5 M NaCl).
  • 1 phage resuspension buffer (1M NaCl, 10 mM TrisHCl pH 7.5, 0.1 mM EDTA).
  • 10 NEB DNase I buffer (10 mM Tris-HCl pH 7.6, 2.5 mM MgCl2, 0.5 mM CaCl2)
  • DNase I (2,000 U/mL) (New England Biolabs M0303)
  • RNase A (20 mg/mL) (Invitrogen PureLink RNase A #12091021)
  • 0.5 M EDTA pH 8.0
  • phenol:chloroform:isoamyl alcohol (25:24:1)
  • 100% and 70% ethanol pre-chilled at 20C.
  • 10 mM Tris HCl pH 7.5

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 and mix well by inverting the tube.
  2. Incubate this mixture for 2 hours to overnight at 4C.
  3. Centrifuge for 30 min at 10,000×g at 4C.
  4. Pour off the supernatant. Resuspend in 360 l of 1 phage resuspension buffer in a 1.7 ml tube.
  5. Add 40 l of 10DNAse I buffer (or to final 1 concentration). Mix by gently vortexing or tapping tube.
  6. Add 1 L DNase I (2000 U/mL) and 1 L RNase A (20 mg/mL). Mix by tapping tube. Incubate for 30 minutes at 37C. This step is very important for degrading any remaining nucleic acids from lysed bacterial cells!
  7. Add 10 l of 0.5 M EDTA (pH 8.0) to chelate divalent metals in the buffer. Mix by gently vortexing or tapping tube.
  8. Phenol-chloroform extract. Add 1 volume (~500 l) of phenol:chloroform:isoamyl alcohol (25:24:1). Shake and invert tube by hand for 15 sec to mix.
  9. Centrifuge at room temperature for 5 minutes at 16,000 g. Carefully remove the aqueous phase on top using a P200 to a new 1.7 ml tube.
  10. Ethanol Precipitate. Add 50 l (1/10 volume) of 3 M sodium acetate. Mix gently. Add 1000 l (2.5 volumes) of 100% ethanol that is pre-chilled to 20C. Keep sample at 20C for at least 30 minutes to overnight.
  11. Centrifuge at 14,000g for 15 minutes at 4C. You should see a pellet. Remove liquid above it.
  12. Add 500 l of 70% ethanol that is pre-chilled at 20C. Pipette up and down to dislodge the pellet form the side of the tube.
  13. Centrifuge at 14,000g for 5 minutes at 4C. Remove all liquid and let air dry.
  14. Resuspend in 50 l of 10 mM Tris HCl pH 7.5 for storage frozen at 20C.
  15. Measure the DNA concentration of in your sample. The T7 genome is double-stranded DNA.

Notes

  • PEG precipitation can be used to concentrate phage stocks. Just stop after resuspending in step 4.
  • 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.

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Contributors to this topic edittopic JeffreyBarrick
Topic revision: r4 - 17 Mar 2020 - 11:40:00 - Main.JeffreyBarrick
 
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