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西亚试剂::DNase I Footprinting

Overview 
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).
 
Procedure
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 ddH2O.

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).

 
 
Solutions
Competitor DNA     Sonicated Calf Thymus DNA in ddH2O
 
 
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 ddH2O
Freeze as 2-5 μl aliquots and store at -70°C
 
 
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 
 
 
 
BioReagents and Chemicals

 
SDS
EDTA
DNase I
HEPES
Polyvinyl Alcohol
Calcium Chloride
Sonicated Calf Thymus DNA
Ethanol
Magnesium Chloride
Chloroform
Phenol
Glycerol
Formamide
Potassium Hydroxide
DTT
Potassium Chloride
Oligonucleotide
Sodium Chloride
Xylene Cyanol FF
Bromophenol Blue
carrier RNA
 
 
Protocol Hints
1. Make a fresh dilution of the DNase I stock solution in ice cold ddH2O and keep on ice. For the no-protein control, use about 1 μl of a 1:1000 dilution. For crude protein extracts, the amount and dilution of the DNase I needs to be determined empirically; 5 μl of a 1:100 dilution is usually adequate. 

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