DNase I Footprinting

Sunday, October 19, 2003

Description
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

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

25 l of protein extract in HEMG

10 l of 10% Polyvinyl Alcohol

1 l of 1 M HEPES, pH 7.6

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

1 to 5 g competitor DNA

and bring the final reaction volume to 50 l 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 l of Ca/Mg Solution to the binding reaction at room temperature.

4. Add 1 to 10 l 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 l of stop solution and mix by vortexing immediately.

7. Add 200 l 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 l formamide dye and load the sample onto a 6% to 8% sequencing gel.

Recipes
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 l aliquots and store at -70C

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 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

Supplies

Tips
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 l of a 1:1000 dilution. For crude protein extracts, the amount and dilution of the DNase I needs to be determined empirically; 5 l of a 1:100 dilution is usually adequate.

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DNase I Footprinting