Protocol F.2

DNaseI Footprintint

 

Solutions

10X Binding Buffer

200 mM Tris 8.0 200 ml 1M Tris pH 8.0

500 mM NaCl 100 ml 5M NaCl

10 mM EDTA 20 ml 0.5 M EDTA pH 8.0

680 ml Q

store at room temperature

 

DNaseI Dilution Buffer

(this buffer provides the Mg+2 for DNaseI activity)

20 mM Tris 7.5 20 ml 1M Tris pH 7.5

50% glycerol 500 ml glycerol

120 mM MgCl2 120 ml 1M MgCl2

360 ml 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 ml DNaseI Storage Buffer:

500 ml 50% Glycerol

50 ml 1M Tris 7.2 @ 25°C 890 ml Tris acid:110 ml Tris base

10 ml 1M MgSO4

2 ml 0.5 M DTT

430 ml Q

Note: make 10 ml Aliq. and store at -70°C, Do Not re-freeze

 

Poly dI:dC & Calf Thymus DNA

for dI:dC make a stock of 1mg/ml and store at -70°C

for Calf Thymus DNA make a stock of 4 mg/ml and store at 4°C

 

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 ml (see table)

ii) 3 ml 10X binding buffer

iii) 2-3 ml dI/dC at 1 mg/ml or 6 mg Calf Thymus DNA

iv) 1 ml probe at 20,000-30,000 cpm/ml (see protocol)

v) 100 mg NE or 100 mg 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 mg/ml).

• Systematically add 1 ml 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 ml 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 ml Q and spin in the microfuge for 10 minutes. Remove 350-400 ml aqueous phase and add 0.1 volume 3M NaOAc 5.2, 1 ml 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 ml formamide loading dye. Boil and load a 7% gel denaturing gel as for sequencing.