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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 |
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- 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)
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- 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).
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- 10× NEB DNase I buffer (10 mM Tris-•HCl pH 7.6, 2.5 mM MgCl2, 0.5 mM CaCl2)
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- DNase I (20 mg/mL)
- RNase A (20 mg/mL)
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- phenol:chloroform:isoamyl alcohol (25:24:1)
- 100% and 70% ethanol pre-chilled at –20°C.
- 10 mM Tris HCl pH 7.5
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Procedure |
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- 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.
- Incubate this mixture for at least 2 h at 4°C
- Centrifuge for 30 min at 10,000×g at 4°C
- Pour off the supernatant and add
- 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!
- Add 0.5 M EDTA (pH 8.0) to chelate divalent metals in the buffer (how much?)
- Phenol-chloroform extract: Add 1 volume of XXXXXXXX
- Ethanol precipitate or run through a PCR cleanup column to exchange the buffer
- Resuspend in 10 mM Tris HCl pH 8.5 for storage.
- Measure the concentration of DNA in your sample. The T7 genome is double-stranded DNA.
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- 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.
- Incubate this mixture for 2 hours to overnight at 4°C.
- Centrifuge for 30 min at 10,000×g at 4°C.
- Pour off the supernatant. Resuspend in 360 µl of 1× phage resuspension buffer in a 1.7 ml tube.
- Add 40 µl of 10×DNAse I buffer (final 1× concentration). Mix by gently vortexing or tapping tube.
- 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!
- 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.
- 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.
- 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.
- 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.
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- Centrifuge at 14,000×g for 15 minutes at 4°C. You should see a pellet. Remove liquid above it.
- 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.
- Centrifuge at 14,000×g for 5 minutes at 4°C. Remove all liquid and let air dry.
- Resuspend in 50 µl of 10 mM Tris HCl pH 7.5 for storage frozen at –20°C.
- Measure the DNA concentration of in your sample. The T7 genome is double-stranded DNA.
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Notes |
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- PEG precipitation can be used to concentrate phage stocks. Just stop after resuspending in step 4.
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- 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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