(r2) Barrick Lab :: ProtocolsPhageGenomicDNA

---+ 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.
   1 Incubate this mixture for at least 2 h at 4°C
   1 Centrifuge for 30 min at 10,000&times;g at 4°C
   1 Pour off the supernatant and add 
   1 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! 
   1 Add 0.5 M EDTA (pH 8.0) to chelate divalent metals in the buffer (how much?)
   1 Phenol-chloroform extract: Add 1 volume of XXXXXXXX
   1 Ethanol precipitate or run through a PCR cleanup column to exchange the buffer
   1 Resuspend in 10 mM Tris HCl pH 8.5 for storage.
   1 [[DNAConcentrationDetermination][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.

Barrick Lab > ProtocolList > ProtocolsPhageGenomicDNA

Contributors to this topic

JeffreyBarrick, VictorLi

Topic revision: r2 - 2020-03-02 - 11:42:02 - Main.JeffreyBarrick