---+ 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 (20 mg/mL) * RNase A (20 mg/mL) * 0.5 M EDTA pH 8.0 * phenol:chloroform:isoamyl alcohol (25:24:1) * 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 1/5 the volume of 5× phage precipitation solution 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×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 (final 1× concentration). Mix by gently vortexing or tapping tube. 1 Add 1 µL DNase I (20 mg/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.
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Topic revision: r3 - 2020-03-02 - JeffreyBarrick