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DNaseI Footprintint【Harvard University】

点击： 作者：51protocol收集 来源： 时间： 2007-03-28 　本站论坛

Cepko/Tabin Lab .Harvard University http://axon.med.harvard.edu/~cepko/protocol/mike/F2.html

Solutions

10X Binding Buffer

200 mM Tris 8.0 200 ml 1M Tris pH 8.0ml 5M NaClml 0.5 M EDTA pH 8.0ml Qstore at room temperature

DNaseI Dilution Buffer

(this buffer provides the Mg 2 for DNaseI activity)

20 mM Tris 7.5 20

50% glycerol 500

120 mM MgCl

ml 1M Tris pH 7.5ml glycerol2 120 ml 1M MgCl2

360

ml Qstore 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

430

ml DNaseI Storage Buffer:ml 50% Glycerolml 1M Tris 7.2 @ 25°C 890 ml Tris acid:110 ml Tris baseml 1M MgSO42 ml 0.5 M DTTml 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 1

for Calf Thymus DNA make a stock of 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

ii) 3

iii) 2-3

iv) 1

v) 100

• 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

• Systematically add 1

• When all the tubes are finished add 500

• 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

mg/ml and store at -70°Cmg/ml and store at 4°Cml (see table)ml 10X binding bufferml dI/dC at 1 mg/ml or 6 mg Calf Thymus DNAml probe at 20,000-30,000 cpm/ml (see protocol)mg NE or 100 mg BSA (see iii)mg/ml).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

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