---+ 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×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.
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Topic revision: r2 - 2020-03-02 - JeffreyBarrick