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  • EpiMark™ Nucleosome Assembly Kit (E5350)

    Introduction

    General Guidelines
    Molar Ratio
    (2 Dimers : 1 Tetramer : 1 DNA Binding Site)
    Because the correct ratio of DNA to protein determines the efficiency of an assembly, the kit components have been formulated to have equal volumes of each added in a reaction. After assembly, the reactions should contain 5–10% unbound DNA to mitigate aggregate formation which occurs when protein is in excess.

    DNA Substrate
    The Nucleosome Control DNA (208 bp) contains one possible binding site for an octamer. This enables visualization by gel shift assay due to the binding of the octamer and a characteristic shift from 208 bp to ~700 bp on a 6% native polyacylamide gel. When using user-supplied substrate DNA, optimization may be required to determine the number of possible binding sites per molecule. A starting point for the amount of user supplied DNA to add per reaction can be determined using the formulas described in the DNA concentration formula section. Then, keeping the amount of DNA constant, the amount of octamer can be varied to find an optimal ratio of DNA to protein. To conserve DNA and protein during optimization, we would recommend the dilution protocol for 25 pmol.

    Other Considerations

    • Because stability can be an issue for the octamer in vitro without DNA, we provide the Dimer and Tetramer separately.
    • Set up each reaction with dH2O, 5 M NaCl, DNA and protein such that the final concentration is 2 M NaCl, noting that the Dimer and Tetramer are supplied in 2 M NaCl containing buffer.
    • Always add the Dimer and Tetramer last!
    • Because nucleosomes can dissociate when too dilute, it is recommended to keep the final protein concentration above 10 µg/ml (5).
    Protocols
    Two different protocols are available. The quickest is the dilution assembly protocol with assembly being ready in less than three hours. It is ideal for optimization since smaller volumes of all components may be used. The dialysis protocol does take longer but the concentration differences from starting material are minimized and it requires less sample handling.

    Protocol

    1. Dilution Assembly Protocol (5)
      Reactions can be scaled up or down depending on the final nucleosome requirement. Methods for 50 pmol and 25 pmol are presented. If the scale is adjusted, it is important to also adjust 5 M NaCl addition such that the starting concentration in the reaction is 2 M NaCl. With sequential dilutions and incubation, the salt concentration is lowered to 0.25 M NaCl, allowing the octamer to bind the DNA and form the nucleosome core particle.

      Materials Required but not Supplied
      5 M NaCl
      Dilution Buffer: 10 mM Tris, pH 8.0
      6% Polyacrylamide gel with gel apparatus and gel buffer (ex: Invitrogen 6% DNA retardation gel)
      100% Glycerol
      TriDye™ 100 bp DNA Ladder (NEB# N3271)
      1X TBE

    2. For 50 pmol
      This reaction, as described, can yield a maximum of 50 pmol nucleosome in 160 µl (0.3 pmol/µl; 0.3 µM nucleosome; 33.7 µg/ml protein).
      1. Place 200 µl of Dilution buffer per reaction at roomtemperature.
      2. Prepare the Reaction Assembly Mix on ice in the following order (for user-supplied substrate, suggested ratios have been included):
            For Optimizing User-supplied
        DNA Substrate
         

        Control
        DNA Only
        (50 pmol)

        0.5 to 1
        Octamer
        to DNA
        1 to 1
        Octamer
        to DNA
        1.5 to 1
        Octamer
        to DNA
        Water 1 μl 0 to 4.5 µl 0 to 7 µl 0 to 1.5 µl
        5M NaCl 4 µl 6 µl 4 µl 2 µl
        DNA 5 μl (10 μM) 50 pmol 50 pmol 50 pmol
        20 μM Dimer 5 μl 2.5 µl 5 μl 7.5 µl
        10 μM Tetramer 5 μl 2.5 µl 5 μl 7.5 µl
        Total 20 µl 20 µl 20 µl 20 µl
        The Dimer and Tetramer are supplied in 2 M NaCl.
      3. Incubate reactions at room temperature for 30 minutes.
      4. Add 7 µl room temperature dilution buffer to each reaction. This brings the reactions to 1.48 M NaCl, 27 µl total volume. Incubate at room temperature for 30 minutes.
      5. Add 13 µl room temperature Dilution Buffer to each reaction. This brings the reactions to 1.0 M NaCl, 40 µl total volume. Incubate at room temperature for 30 minutes.
      6. Add 27 µl room temperature Dilution Buffer to each reaction. This brings the reactions to 0.6 M NaCl, 67 µl total volume. Incubate at room temperature for 30 minutes.
      7. Add 54 µl room temperature Dilution Buffer to each reaction. This brings the reactions to 0.25 M NaCl, 160 µl total volume. Incubate at room temperature for 30 minutes.
      8. Store samples at 4°C.
      9. Use gel shift assay to analyze samples.
    3. For 25 pmol
      This reaction can yield a maximum of 25 pmol nucleosome in 80 µl (0.3 pmol/µl; 0.3 µM nucleosome; 33.7 µg/ml protein).
      1. Place 100 µl of Dilution Buffer per reaction at room temperature.
      2. Prepare the Reaction Assembly Mix on ice in the following order (for user-supplied substrate, suggested ratios have been included):
            For Optimizing User-supplied
        DNA Substrate
         

        Control
        DNA Only
        (25 pmol)

        0.5 to 1
        Octamer
        to DNA
        1 to 1
        Octamer
        to DNA
        1.5 to 1
        Octamer
        to DNA
        Water 0.5 μl 0 to 4.5 µl 0 to 7 µl 0 to 1.5 µl
        5M NaCl 2 µl 3 µl 2 µl 1 µl
        DNA 2.5 μl (10 μM) 25 pmol 25 pmol 25 pmol
        20 μM Dimer 2.5 μl 1.25 µl 2.5 μl 3.75 µl
        10 μM Tetramer 2.5 μl 1.25 µl 2.5 μl 3.75 µl
        Total 10 µl 10 µl 10 µl 10 µl
        The Dimer and Tetramer are supplied in 2 M NaCl.
      3. Incubate reactions at room temperature for 30 minutes.
      4. Add 3.5 µl room temperature dilution buffer to each reaction. This brings the reactions to 1.48 M NaCl, 13.5 µl total volume. Incubate at room temperature for 30 minutes.
      5. Add 6.5 µl room temperature Dilution Buffer to each reaction. This brings the reactions to 1.0 M NaCl, 20 µl total volume. Incubate at room temperature for 30 minutes.
      6. Add 13.5 µl room temperature Dilution Buffer to each reaction. This brings the reactions to 0.6 M NaCl, 33.5 µl total volume. Incubate at room temperature for 30 minutes.
      7. Add 46.5 µl room temperature Dilution Buffer to each reaction. This brings the reactions to 0.25 M NaCl, 80 µl total volume. Incubate at room temperature for 30 minutes.
      8. Store samples at 4°C.
      9. Use gel shift assay to analyze samples.
    4. Dialysis Assembly Protocol (5)
      This reaction can yield a maximum of 50 pmol nucleosome in ~150 µl or 33 nM nucleosome; 36 µg/ml protein. It can be increased in scale if more material is required. The starting salt concentration is 2 M NaCl. With sequential dialysis over time, the salt concentration is lowered to 0.25 M NaCl forming the nucleosome core particle.Place 200 µl of Dilution buffer per reaction at roomtemperature.

      Materials Required but not Supplied
      5 M NaCl

      Dialysis units (like Pierce Slide-a-lyzer mini dialysis units 10,000 MWCO)

      Dialysis buffers:
      • 20 mM Tris-HCl, pH 8.0, 1.5 M NaCl, 1 mM EDTA, 1 mM DTT
      • 20 mM Tris-HCl, pH 8.0, 1.0 M NaCl, 1 mM EDTA, 1 mM DTT
      • 20 mM Tris-HCl, pH 8.0, 0.6 M NaCl, 1 mM EDTA, 1 mM DTT
      • 20 mM Tris-HCl, pH 8.0, 0.25 M NaCl, 1 mM EDTA, 1 mM DTT
      6% Polyacrylamide gel with gel apparatus and gel buffer (ex: Invitrogen 6% DNA retardation gel)
      100% Glycerol
      TriDye™ 100 bp DNA Ladder (NEB #N3271)
      1X TBE
      1. Prepare 0.5 L of each of the four dialysis buffers and chill to 4°C.
      2. Prepare each reaction on ice in the following order and mix well:
            For Optimizing User-supplied
        DNA Substrate
         

        Control
        DNA

        0.5 to 1 1 to 1 1.5 to 1
        Water 49 μl 0 to 57 µl 0 to 54 µl 0 to 48 µl
        5M NaCl 36 µl 38 µl 36 µl 32 µl
        DNA 5 μl (10 μM) 50 pmol 50 pmol 50 pmol
        20 μM Dimer 5 μl 2.5 µl 5 μl 10 µl
        10 μM Tetramer 5 μl 2.5 µl 5 μl 10 µl
        Total 100 µl 100 µl 100 µl 100 µl
        The Dimer and Tetramer are supplied in 2 M NaCl.
      3. Transfer the reaction to the mini dialysis units according to the manufacturers protocol.
      4. Place dialysis units in 1.5 M NaCl buffer for 2–3 hours at 4°C and then transfer to each consecutively lower NaCl concentration buffer for 2–3 hours at 4°C with either the 0.6 M or 0.25 M NaCl buffer dialysis being an overnight step.
      5. Transfer the samples to tubes. The volume will have increased because of the salt dialysis. Equalize sample volumes to 150 µl using the 0.25 M NaCl buffer. If volumes are off by more than 20%, some thing may have gone wrong with the set up of those samples.
      6. Store samples at 4°C.
      7. Use gel shift assay to analyze samples.