---+ 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×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
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Topic revision: r1 - 2020-02-29 - JeffreyBarrick