DNase I genomic footprinting of salivary glands permeabilized with NP40.

The following genomic footprinting protocol was developed by Hongbing Tang.  It has been used successfully but not extensively.  One change that might be worth evaluating is the addition of CaCl2 (final concentration: 0.5 mM) to the glands before adding DNase I.  This will increase the DNase I activity by approximately 10-fold.  It is also likely that one or two hours of digestion with proteinase K will suffice during the purifiection of the DNA.  Finally, the most problematic part of the whole procedure is often the preparation of the "no protein" control.  This requires purified, intact genomic DNA that has been treated with a range of DNase I conditions.  Plan on testing many samples to find some that match well with the extent of digestion observed in the glands.
  1. Add 1 ul 50 mM DTT to 100ul buffer M (10 mM Hepes pH 7.6, 25 mM KCl, 5 mM MgCl2, 5% glycerol, 0.1mM EGTA) right before use, place the tube on ice.
  2. Dissect 6-8 pairs of salivary glands from 3rd instar larvae and transfer to the tube with M buffer.
  3. Add 2.5% of 20% NP-40, flick the tube gentlely to mix well. Leave on ice for 15 min, flick the tube every 3 minutes.
  4. Dilute DNase I (Worthington, DPRF grade, made up to 20 U/ul) to 1U/ul in buffer M (minus EGTA). Store on ice.
  5. Place the tube of glands at room temperature for 1 min.
  6. Add 4 ul to 8 ul diluted DNase I to the tube and mix well. Incubate at room temperature for 2 minutes.
  7. Add 2 ul 0.5 M EDTA to the tube to inhibit the DNase I, mix well, then add 100 ul of S buffer (20 mM Tris pH 7.4, 200 mM NaCl, 2% SDS, 200 ug/ml proteinase K). Incubate at 37oC overnight.
  8. Extract three times with phenol/chloroform/isoamyl alcohol (49.5:49.5:1).  Ethanol precipitate in the standard way by adding 1/10 volume of 3M sodium acetate (pH ~6) and 2.5 volumes of ethanol.  Wash the ethanol precipitate with cold 70% ethanol. Dissolve final nucleic acid precipitate in 22 ul TE.
  9. Use 1ul of sample to determine DNA concentration. Run 1ul on agarose gel to check for digestion.  Samples treated with DNase I should exhibit a broad smear whereas untreated samples should appear as a high molecular weight band.
  10. Analyze 100 ng of DNA by LM-PCR.