Home Protocols Dialysis Assembly Protocol (E5350)

Dialysis Assembly Protocol (E5350)

Introduction

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

    Protocol

    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 7.5 µl
      10 μM Tetramer 5 μl 2.5 µl 5 μl 7.5 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.