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  • Dilution Assembly Protocol (E5350)

    Introduction

    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

    Protocol

    1. 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 93 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.
    2. 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.