Dialysis Assembly Protocol (E5350)
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)
TriDye™ 100 bp DNA Ladder (NEB#N3271 )
- Prepare 0.5 L of each of the four dialysis buffers and chill to 4°C.
- Prepare each reaction on ice in the following order and mix well:
For Optimizing User-supplied DNA Substrate
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
- Transfer the reaction to the mini dialysis units according to the manufacturers protocol.
- 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.
- 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.
- Store samples at 4°C.
- Use gel shift assay to analyze samples.