ProtocolsPhageGenomicDNA < Lab

---+ 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 Tris•HCl pH 7.5, 0.1 mM EDTA).
   * 10× NEB DNase I buffer (10 mM Tris-•HCl pH 7.6, 2.5 mM MgCl<sub>2</sub>, 0.5 mM CaCl<sub>2</sub>)
   * 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)
   * 3 M sodium acetate
   * 100% and 70% ethanol pre-chilled at –20°C.
   * 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 5× phage precipitation solution equal to 1/4 the volume of phage lysate and mix well by inverting the tube.
   1 Incubate this mixture for 2 hours to overnight at 4°C.
   1 Centrifuge for 30 min at 10,000&times;g at 4°C.
   1 Pour off the supernatant. Resuspend in 360 µl of 1× phage resuspension buffer in a 1.7 ml tube.
   1 Add 40 µl of 10×DNAse I buffer (or to final 1× concentration). Mix by gently vortexing or tapping tube.
   1 Add 1 µL DNase I (2000 U/mL) and 1 µL RNase A (20 mg/mL). Mix by tapping tube. Incubate for 30 minutes at 37°C. This step is very important for degrading any remaining nucleic acids from lysed bacterial cells! 
   1 Add 10 µl of 0.5 M EDTA (pH 8.0) to chelate divalent metals in the buffer. Mix by gently vortexing or tapping tube.
   1 Phenol-chloroform extract. Add 1 volume (~500 µl) of phenol:chloroform:isoamyl alcohol (25:24:1). Shake and invert tube by hand for 15 sec to mix. 
   1 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.
   1 [[ProtocolsEthanolPrecipitation][Ethanol Precipitate]]. Add 50 µl (1/10 volume) of 3 M sodium acetate. Mix gently. Add 1000 µl (2.5 volumes) of 100% ethanol that is pre-chilled to –20°C. Keep sample at –20°C for at least 30 minutes to overnight. 
   1 Centrifuge at 14,000×g for 15 minutes at 4°C. You should see a pellet. Remove liquid above it.
   1 Add 500 µl of 70% ethanol that is pre-chilled at –20°C. Pipette up and down to dislodge the pellet form the side of the tube.
   1 Centrifuge at 14,000×g for 5 minutes at 4°C. Remove all liquid and let air dry.
   1 Resuspend in 50 µl of 10 mM Tris HCl pH 7.5 for storage frozen at –20°C.
   1 [[DNAConcentrationDetermination][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.
Barrick Lab > ProtocolList > ProtocolsPhageGenomicDNA
Topic revision: r6 - 2021-06-16 - 21:03:35 - Main.VictorLi