DNase I Footprinting
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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
