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.