Protocol for High Molecular Weight DNA (HMW DNA) Extraction from Phage

Below is a detailed protocol for the extraction of high molecular weight DNA from phage samples using the Monarch HMW DNA Extraction Kit for Tissue (NEB #T3060).

Materials Required but Not Supplied

  • DNase I, 20 mg/ml. Prepare 20 mg/ml stock by dissolving DNase I (such as DNase I, cat# LS002004, from Worthington Biochemical or another equivalent) in DNase I storage buffer (10 mM Tris pH 7.6, 2 mM CaCl2, and 50% glycerol).
  • 3X precipitant solution (30% w/v PEG-8000, 3 M NaCl); can be omitted if alternative pelleting approach is employed in Part 1: Step 3.
  • Resuspension solution (5 mM MgSO4 in water)
  • Isopropanol, 550 µl per sample
  • Ethanol (≥ 95%)
  • Thermal mixer containing a 2 ml tube block (if not available, use a 1.5 ml tube block).
  • 50 ml centrifuge tubes
  • Syringe of appropriate size, sterile filter (0.45 or 0.22 μm)
  • 1.5 ml microfuge tube (1 per sample)
  • Recommended: vertical rotating mixer (e.g., Thermo Scientific® HulaMixer® Sample Mixer)
  • Wide-bore pipette tips
  • Microcentrifuge

Important Notes Before You Begin

  • Store Proteinase K and RNase A at -20°C upon opening
  • Review the complete protocol before beginning
  • Prepare a nuclease solution by combining 1 volume of DNase I stock (user-supplied, 20 mg/ml) with 1 volume of RNase A enzyme solution (included, 20 mg/ml), so that the final concentration of the combined nuclease solution is 10 mg/ml. Prepare a sufficient total volume of nuclease solution to process all samples: each sample will require 1 µl of nuclease solution per ml of lysate.
  • Add ethanol (≥ 95%) to the gDNA Wash Buffer as indicated on the bottle label
  • Preheat thermal mixer with 2.0 ml tube block (or 1.5 ml tube block if not available) to 56°C

Starting Material Notes

Starting material is 10-50 ml phage lysate with >109 PFU/ml. As a rule of thumb, 1 ml of phage lysate with a titer of 1 x 1010 contains about 0.5 μg of phage DNA, assuming the phage has a 50 kb genome. Calculate input culture volume so that a yield of 10 -100 µg phage DNA can be expected. Typically, 10-50 ml of a high-titer phage lysate is used for DNA extraction.

HMW gDNA Purification Consists of Two Stages

PART 1: Phage Collection and Lysis

PART 2: HMW gDNA Binding and Elution

PART 1: Phage Collection and Lysis

  1. Prepare phage lysate by plate lysate or liquid culture methods. Lysates should be clarified by filter sterilization (0.45 or 0.22 μm). Lysates may be in broth (LB, TSB, etc.) or in λ-diluent.

  2. Place the lysate into a clean 50 ml centrifuge tube and add 1 μl of nuclease solution per ml of lysate. The nuclease solution is a combined solution of DNase I & RNase A at a final concentration of 10 mg/ml. Incubate at 37ºC for 30 minutes, or at room temperature for 2 hours.

  3. Add 3X precipitant solution to the lysate at a ratio of 1:2 precipitant:lysate (10% PEG-8000, 1 M NaCl final concentration). Mix gently by inversion. Incubate on ice for at least 60 minutes. Optimal precipitation occurs when incubated at 4ºC overnight. Most phages are stable in this state for up to several days. 

    Alternative method: phages may be pelleted from the lysate by centrifugation in a normal high-speed centrifuge (e.g., about 7,000 x g overnight) or in an ultracentrifuge (e.g., 50,000 x g for about 1 hour). dsDNA phages weigh about 400-1000 S, and pelleting time (in hours) can be calculated from the rotor’s k-factor by the formula: time = k/S.

  4. Centrifuge the precipitated phage lysate at 10,000 x g at 4ºC for 10 minutes. Carefully pour off the supernatant and retain the pellet. The pellet may be transparent or opaque and may be spread up the wall of the tube.

  5. Resuspend the pellet in 300 μl of resuspension buffer (5 mM MgSO4) by pipetting gently up and down; be sure to rinse the sides of the tube to obtain all of the pellet.

  6. Transfer the resuspended phage to a labeled 1.5 ml microcentrifuge tube and centrifuge for 5-10 seconds in a benchtop minicentrifuge to pellet any insoluble particles. Transfer the supernatant to a new labeled Monarch 2 ml Tube. 

  7. Add 300 µl HMW gDNA Tissue Lysis Buffer and 20 µl Proteinase K. Incubate samples in a thermal mixer at 56°C for a minimum of 45 minutes with agitation at 500 rpm. If a thermal mixer is not available use a heat block and mix every 10-15 minutes by inverting the tube 5 times.

  8. Add 300 µl Protein Separation Solution and mix by inverting for 1 minute. Alternatively, a vertical rotating mixer at 20 rpm can be used.

  9. Centrifuge for 10 minutes at 16,000 x g. If working with multiple samples, during centrifugation, prepare the plastics for Part 2, as indicated in the following step. The sample will separate into a large, clear phase (DNA) and a smaller protein phase, usually on the bottom of the tube, but occasionally the protein phase may be floating as little hydrophobic bubbles on the surface of the solution. 

  10. If working with multiple samples, prepare and label the plastics for the upcoming steps. Each sample will require:

    • 1 Monarch Collection Tube II (no need to label)
    • 1 Monarch Bead Retainer inserted into the collection tube; this will be used to remove the wash buffer from the gDNA bound to the beads.
    • 2 Monarch 2 ml Tubes; one for phase separation and one for elution.
    • 1 1.5 ml microfuge tube (DNA low bind recommended, not provided); this will be used to collect the eluate.

  11. Using a 1000 µl wide-bore pipette tip, transfer the upper phase containing the DNA (large, clear phase) to a labeled Monarch 2 ml Tube. Highest yields will be achieved by transferring as much of the upper phase as possible. We recommend using a 200 µl wide-bore pipette tip to transfer the final volume of the upper phase for maximum yield. Avoid transferring material from the protein layer, although a small amount (1–2 µl) will not be detrimental. If a small amount of the protein phase enters the pipette tip, gently push it back into the tube. If a lower protein phase is not visible, leave ~30 µl behind to ensure protein is not carried over. Typically, the transferred volume will be ~ 800 µl. 

PART 2: Phage DNA Binding and Elution

  1. Using clean forceps, add 2 DNA Capture Beads to each sample, which should be contained in a Monarch 2 ml Tube.

  2. Add 550 µl isopropanol, close the cap, and mix on a vertical rotating mixer at 10 rpm for 4 minutes to attach DNA to the beads. If a vertical rotating mixer is not available, invert slowly and gently by hand 25–30 times. A manual inversion is complete when the tube returns to the upright position. Slow inversion is critical for the DNA to bind to the beads; each full inversion should take ~5–6 seconds. If necessary, flick the tube to release any beads that stick to the bottom of the tube.

  3. Remove and discard liquid by pipetting. Avoid removing any of the DNA wrapped around the glass beads. For optimal DNA solubility, avoid letting the bound DNA dry out on the beads during this and the following steps; add the next buffer quickly. There are two suggested options for carrying out this step: 

    • Keeping tube upright, insert pipette tip and gently push beads aside to remove liquid. 
    • Angle the tube so that beads remain at the bottom, and liquid reaches toward tube opening. Pipette from the liquid surface and continue to angle as liquid is removed (tube will be almost horizontal at the end).

  4. Add 500 µl gDNA Wash Buffer, close the cap, and mix by inverting the tube 2–3 times. Remove the gDNA Wash Buffer as described in Step 3. The loose gDNA complex will condense around the beads more tightly. 

  5. Repeat the wash in Step 4 and remove the gDNA Wash Buffer by pipetting.  Alternatively, the buffer can be removed by decanting: position a pipette tip at the top of the angled tube to prevent the beads from falling out. It is not necessary to remove all the gDNA Wash Buffer at this point.

  6. Place a labeled bead retainer into a Monarch Collection Tube II. Pour the beads into the bead retainer and close the cap. Discard the used Monarch 2 ml Tube. When working with multiple samples, be sure to close the cap of the bead retainer after each transfer of beads. 

  7. Pulse spin (≤ 1 second) the sample in a benchtop minicentrifuge to remove any residual wash buffer from the beads.

  8. Separate the bead retainer from the collection tube, pour the beads into a new, labeled Monarch 2 ml Tube, and insert the used bead retainer into the labeled 1.5 ml microfuge tube (DNA low bind recommended, not provided) for later use during elution. Discard the used collection tube.

  9. Immediately add 100 µl Elution Buffer II to the glass beads and incubate for a minimum of 5 minutes at 56°C in a thermal mixer with agitation at the lowest speed (300 rpm). Halfway through the incubation, ensure the beads are not stuck to the bottom of the tube by tilting the tube almost horizontally and gently shaking. This ensures that the beads can move freely, allowing for optimal release of the DNA from the beads. It also ensures that the lower bead does not stick to the bottom of the tube during the following transfer step. Elution volume can be reduced to as low as 50 μl without affecting recovery. However, if using < 100 μl, the gentle shaking of the sample should be done several times during the incubation to ensure complete wetting of the beads.

  10. Ensure the bead retainer is inserted into the 1.5 ml microfuge tube. Pour the eluate and the glass beads into the bead retainer and close the cap. When working with more than 1 sample, it is important to close the cap after each transfer of beads. Typically, all the eluate flows into the bead retainer upon pouring. If any volume remains in the 2 ml tube, spin briefly and transfer.

  11. Centrifuge for 30 seconds at 12,000 x g to separate the eluate from the glass beads. Discard the beads and retainer. 

  12. Pipette eluate up and down 5–10 times with a wide bore pipette tip and ensure any visible DNA aggregates are dispersed. Before analysis or downstream use, the DNA must be homogeneously dissolved. After pipetting, incubate at 37°C for 30-60 minutes, overnight at room temperature, or for > 24 hours at 4°C. Pipette up and down 5-10 times again before analyzing or using the phage DNA. The elution buffer (10 mM Tris, pH 9.0, 0.5 mM EDTA) is formulated for long term storage of DNA.

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