Set up binding reaction (standard reaction is in 25 ul, don't forget a no-protein control):

1. 5 ul of 50 mM HEPES pH 7.6, 25 mM MgCl₂.
2. 1 ul of 1 ug/ul HaeIII-cut E.coli DNA (nonspecific DNA to limit nonspecific binding).
3. 0.125 ul of 100 mM DTT.
4. 15,000 cpm of probe (generally in a volume of <1ul of 10 mM Tris pH 8, 1 mM EDTA).
5. An amount of 1M KCl so that the final KCl concentration, including that contributed by the protein solution, will be 80 to 100 mM.
6. An amount of H₂O so that the final volume of the reaction will be 25 ul after everything including protein has been added.
7. An amount of 25 mM HEPES pH 7.6, 10% glycerol, 0.1 mM EDTA so that this in combination with the protein totals 10 ul.
8. Mix and briefly centrifuge so that all of the solution is at the bottom.
9. Place tubes in a float in a 25^oC water bath. This bath can be set up in a styrofoam box.

DNase 1 solution:

1. Stagger addition of protein to each tube by 1 minute intervals. Mix each addition thoroughly by triterating the mixture with the pipette. Avoid generating bubbles.
2. Return each sample to the 25^oC water bath and then proceed to the next sample.
3. One sample should be a no protein control. Buffer matching that of the protein samples should be added to the binding reaction.

1. After 30' of binding, add 2 ul of DNAse 1 solution. Thoroughly mix by triterating for a few seconds.
2. Return the sample to the 25^oC water bath.
3. 40 seconds after addition of DNase 1, stop digestion by adding 25 ul of Proteinase K stop.
4. Proceed with each subsequent DNase 1 digestion, each being staggered by 1'.

1. Proteinase K treat sample at 37^oC for at least 15'. Now is a good time to set up the sequencing gel if you want to run the gel immediately after processing the DNA.
2. Add 100 ul of chloroform/phenol mix and shake vigorously for about 1'. Centrifuge for 4' at 20^oC. Remove the organic phase by penetrating the aqueous phase with a yellow tip. Draw out most of the organic phase, leaving behind 1-2 ul of the organic along with all of the aqueous.
3. Add 5 ul of 3 M Na Acetate pH 5-6 and 125 ul of ethanol, mix by inverting and shaking the tube.
4. Chill on ice for at least 15'
5. Centrifuge for at least 20'
6. Remove supernatant from each tube and return the tube to the ice. After removing the supernatant from all of the tubes, go back and remove trace amounts of supernatant with a drawn glass pipette.
7. Air dry the precipitate or place it in the vacuum jar for about 10'.
8. Dissolve the precipitate in 5 ul of sequencing load buffer being sure to rap the tube so that load buffer comes in contact with all of the lower half of the tube. Give the sample at least 10' to dissolve.
9. Store the samples at -20^oC until they are to be run on a sequencing gel. Just prior to loading the samples on the gel, boil them for 5' and then quench them on ice. Don't forget this.

• The digestions can be fine tuned by changing the length of the DNase 1 digestion time. For example, the no protein control is often more sensitive to DNase 1 so its digestion time might be shortened to 30 seconds whereas a sample with lots of protein might be lengthened to 50 seconds
• Changing the concentration of nucleic acid requires a change in the amount of DNase 1. For example, the exonuclease III binding conditions will require 2 to 3 times more DNase 1
• Binding reactions can be performed on ice, in the absence of MgCl₂, or both if endogenous nuclease activities are unacceptably high. Special considerations must be made in making up the DNase 1 dilutions. The following is a guide but preliminary tests with varying amounts of DNase 1 would should be tested to optimize the footprinting reactions.
• Binding at 25^oC in the absence of MgCl₂: dilute DNase 1 to 0.05 U/ul in 50 mM NaCl, 20 mM Tris pH 7.6, 15 mM MgCl₂, 3 mM CaCl₂ and use 4 ul on a 25 ul binding mix for 40 seconds at 25^oC
• Binding at 0^oC in the absence of MgCl₂: dilute DNase 1 to 0.5 U/ul in 50 mM NaCl, 20 mM Tris pH 7.6, 15 mM MgCl₂, 3 mM CaCl₂ and use 4 ul on a 25 ul binding mix for 40 seconds at 0^oC
• Binding at 0^oC in the presence of MgCl₂: dilute DNase 1 to 0.5 U/ul in 50 mM NaCl, 20 mM Tris pH 7.6, 5 mM MgCl₂, 3 mM CaCl₂ and use 4 ul on a 25 ul binding mix for 40 seconds at 0^oC

I've altered the DNase 1 footprinting in two ways:

1. Include acetylated BSA at 100 ug/ml in all binding reactions, even the no protein control. This should help stabilize proteins at low concentrations when they are purified and also even out the DNase 1 digestions for samples that would otherwise have significantly protein concentrations.
2. Include CaCl₂ at a final concentration of 0.5 mM during the digestion. The CaCl₂ is added after the binding reaction and is part of the solution containing the DNAse 1. The CaCl₂ increases the DNase 1 activity by about 10-fold. The lower level of DNase 1 that is required for digestion might reduce the likelihood that the DNase 1 displaces a DNA binding protein.

Incubate at 25^oC for a suitable length of time, usually around 30'.

Prepare a fresh dilution of DNase 1 to 0.01 Units/ul in ice cold 50 mM NaCl, 20 mM Tris pH 7.5, 5 mM MgCl₂, 5 mM CaCl₂, 100 ug/ml acetylated BSA. Do this by serial dilution: first dilute a small amount of DNase 1 from the stock to 1 U/ul and then dilute this first dilution to 0.15 U/ul. Gently triterate at each step to mix the DNase 1 (vortexing will inactivate the DNase 1).

For digestion: add 2.5 ul of the dilute DNase 1 to the sample, briefly triterate and return to 25^oC bath for 40 seconds. Stop digestion with 25 ul of 0.5% Sarkosyl, 10 mM EDTA, 100 ug/ml salmon DNA, 50 ug/ml proteinase K. Incubate at 37^oC for 15' and proceed with organic extractions and ethanol precipitations.

Note: you may have to tune-up the concentration of DNase 1 but make sure that the amount added to the binding reaction is 1/10 of the volume of the binding reaction if you want the CaCl₂ concentration be ~0.5 mM during the digestion.