西亚试剂：：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

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