(r1) Barrick Lab :: ProtocolsPhageGenomicDNA

---+ 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.
   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 to degrade any remaining bacterial nucleic acids. 
   1 (Add EDTA to soak up whatever is in the buffer)
   1 Add 50 µg/mL Proteinase K and incubate at 65°C.
   1 Phenol-chloroform extract
   1 Ethanol precipitate
   1 Resuspended in 10 mM Tris HCl pH 8.5 for analysis

Barrick Lab > ProtocolList > ProtocolsPhageGenomicDNA

Contributors to this topic

JeffreyBarrick, VictorLi

Topic revision: r1 - 2020-02-29 - 17:15:51 - Main.JeffreyBarrick