DNase?I?Footprinting

 An end labeled DNA probe is incubated with a purified DNA-binding factor or with a protein extract. The unprotected DNA is then digested with DNase I such that on average, every DNA molecule is cut once. Digestion products are then resolved by electrophoresis. Comparison of the DNase I digestion pattern in the presence or absence of protein will allow the identification of a footprint (protected region).

1. Make the DNA binding reaction by combining the following components in a microcentrifuge tube:

25 μl of protein extract in HEMG

10 μl of 10% Polyvinyl Alcohol

1 μl of 1 M HEPES, pH 7.6

2 fmol of end-labeled DNA probe (about 1200 cpm)

1 to 5 μg competitor DNA

and bring the final reaction volume to 50 μl with ddH₂ O.

2. Mix the components gently and incubate on ice for 10 min (for incubations with purified proteins, incubation temperatures may be increased).

3. Add 50 μl of Ca/Mg Solution to the binding reaction at room temperature.

4. Add 1 to 10 μl of DNase I solution (see Hint #1).

5. Mix quickly and incubate at room temperature for 1 min.

6. Stop the reaction by adding 100 μl of stop solution and mix by vortexing immediately.

7. Add 200 μl of phenol/chloroform and mix by inverting several times. Centrifuge at full speed in a microcentrifuge for 10 min to separate the phases. Recover the aqueous phase and transfer it to a fresh microcentrifuge tube.

8. Add 1 ml of 100% ethanol and mix by inverting several times.

9. Centrifuge at full speed in a microcentrifuge for 10 min to pellet the DNA and remove the ethanol.

10. Resuspend the pellet in 70% ethanol and pellet the DNA again. Remove the ethanol and allow the DNA pellet to air dry.

11. Resuspend the DNA in 6 μl formamide dye and load the sample onto a 6% to 8% sequencing gel (see Protocol on Sequencing Gel Protocol). 

Competitor DNA		Sonicated Calf Thymus DNA in ddH2 O
Formamide Dye		0.005% (w/v) Xylene Cyanol FF 20 mM EDTA make up in deionized Formamide 0.005% (w/v) Bromophenol Blue
70% (v/v) Ethanol
1:1 Phenol:Chloroform
Stop Solution		1% (w/v) SDS 0.2 M NaCl Store at room temperature 20 mM EDTA, pH 8.0 0.25 mg/ml carrier RNA
Ca/Mg Solution		5 mM CaCl2 10 mM MgCl2
DNase I		2.5 mg/ml in ddH2 O Freeze as 2-5 μl aliquots and store at -70℃
HEMG		0.1 mM EDTA, pH 8.0 0.1 M KCl 25 mM HEPES, pH 7.6 with KOH 12.5 mM MgCl2 10% (v/v) Glycerol 1 mM DTT (add just before use)
1 M HEPES		pH 7.6 with KOH
10% (v/v) Polyvinyl Alcohol

Competitor DNA      Sonicated Calf Thymus DNA in ddH₂ O

Formamide Dye      0.005% (w/v) Xylene Cyanol FF

20 mM EDTA

make up in deionized Formamide

0.005% (w/v) Bromophenol Blue

70% (v/v) Ethanol

1:1 Phenol:Chloroform

Stop Solution      1% (w/v) SDS

0.2 M NaCl

Store at room temperature

20 mM EDTA, pH 8.0

0.25 mg/ml carrier RNA

Ca/Mg Solution      5 mM CaCl₂

10 mM MgCl₂

DNase I      2.5 mg/ml in ddH₂ O

Freeze as 2-5 μl aliquots and store at -70℃

HEMG      0.1 mM EDTA, pH 8.0

0.1 M KCl

25 mM HEPES, pH 7.6 with KOH

12.5 mM MgCl₂

10% (v/v) Glycerol

1 mM DTT (add just before use)

1 M HEPES      pH 7.6 with KOH

10% (v/v) Polyvinyl Alcohol