DNaseI Foot printint
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DNaseI Footprintint
Solutions
10X Binding Buffer
200 mM Tris 8.0 200 m l 1M Tris pH 8.0
500 mM NaCl 100 m l 5M NaCl
10 mM EDTA 20 m l 0.5 M EDTA pH 8.0
680 m l Q
store at room temperature
DNaseI Dilution Buffer
(this buffer provides the Mg 2 for DNaseI activity)
20 mM Tris 7.5 20 m l 1M Tris pH 7.5
50% glycerol 500 m l glycerol
120 mM MgCl2 120 m l 1M MgCl 2
360 m l Q
store at room temperature
DNaseI Stocks
I have found that the low grade DNaseI is sufficient and there is no reason to use the RQ DNaseI.
5 mg DNaseI Type II (Sigma Cat.# D4527: 20,000 units)
500 m l DNaseI Storage Buffer:
500 m l 50% Glycerol
50 m l 1M Tris 7.2 @ 25℃ 890 m l Tris acid:110 m l Tris base
10 m l 1M MgSO4
2 m l 0.5 M DTT
430 m l Q
Note: make 10 m l Aliq. and store at -70℃, Do Not re-freeze
Poly dI:dC & Calf Thymus DNA
for dI:dC make a stock of 1m g/ m l and store at -70℃
for Calf Thymus DNA make a stock of 4 m g/ m l and store at 4℃
Procedure
• Generate a table of binding reaction parameters and DNaseI concentrations. All reactions must be in triplicate and a BSA control must be included. It is often wise to include a (-DNaseI) lane as well; this serves as a marker for the relative amount of intact probe remaining and to ensure that there is no nonspecific degradation of the probe.
• Mix the ingredients of the reaction in the following order:
i) Q up to 30 m l (see table)
ii) 3 m l 10X binding buffer
iii) 2-3 m l dI/dC at 1 m g/ m l or 6 m g Calf Thymus DNA
iv) 1 m l probe at 20,000-30,000 cpm/ m l (see protocol)
v) 100 m g NE or 100 m g BSA (see iii)
• Incubate at room temperature for 30 minutes. During the last 5 minutes, prepare the DNaseI dilutions in DNaseI Dilution Buffer (concentrations vary: 0.125-5 m g/ m l).
• Systematically add 1 m l DNaseI dilution (t=0) and vortex on 4 until t=10 seconds and move on to the next tube by t=20 seconds. With this type of stagger, 6 tubes can be processed an once. As the last tube is finished with DNaseI addition go back to the first tube (t= 2 minutes) and add 500 m l phenol/chloroform and vortex on 10 until t=2 minutes 10 seconds and move onto the next tube by t=2 minutes 20 seconds.
• When all the tubes are finished add 500 m l Q and spin in the microfuge for 10 minutes. Remove 350-400 m l aqueous phase and add 0.1 volume 3M NaOAc 5.2, 1 m l tRNA and 2 volumes EtOH.
• Spin in the microfuge for 15 minutes, aspirate most of the EtOH and get the last bit by hand. Dry in the speedvac and resuspend in 6 m l formamide loading dye. Boil and load a 7% gel denaturing gel as for sequencing.
