0.1% Sodium Deoxycholate
1mM EDTA
ChIP lysis buffer (High Salt)(filter sterilize,store at 4 degrees C)
50 mM HEPES pH 7.5
500 mM NaCl
1% TritonX-100
0.1% Sodium Deoxycholate
1 mM EDTA
ChIP wash buffer (filter sterilize,store at 4 degrees C)
10 mM Tris pH 8.0
250 mmliCl
0.5% NP-40
0.5% Sodium Deoxycholate
1 mM EDTA
1x TE pH 8.0 (filter sterilize,store at 4 degrees C)
ChIP elution buffer (store at room temperature)
50 mM Tris pH 8.0
1% SDS
10 mM EDTA
Protease inhibitors
PMSF,Benzamidine,Pepstatin,Leupeptin,and Chymostatin
Phosphatase inhibitors (store at �20 degrees C)
Solution A (100x)for 10 ml
100 mM sodium pyrophosphate 0.45g
100 mM sodium orthovanadate 0.18g
Water to 10 ml
Solution B (100x)for 10ml
100 mM beta-glycerophosphate 0.29g
100 mM EGTA 0.38g
1M NaF 0.42g
Water to 10 ml
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Notes on ChIP method:
Day 2
Step 1.A dilution of 1:1000 of a saturated culture of SHy278 will grow to approximately OD600 1.0 in 16 hours when grown at 30 degrees C.
Day 3
Step 4.The duration of formaldehyde treatment is a point of optimization.Shorter treatments will generally yield higher concentrations of soluble protein,but less extensive cross-linking of proteins to DNA.Longer treatments will yield the opposite,and consequently there will be less material in the extracts for doing IP’s.However,this is not the first step that should be optimized.20 minutes is probably a good starting point for this step unless the effect being observed is expected to occur in a very short time frame.In that case 5-10 minutes formaldehyde treatment may be more appropriate.Optimization of this step involves maximizing the IP/IN ratio for the protein-DNA interaction being assayed,and keeping the duration of formaldehyde treatment short.This can be examined by PCR after IP conditions have been optimized by Western analysis.
Day 4
Step 1.If assaying for phosphorylated proteins,add phosphatase inhibitors at this and subsequent steps when protease inhibitors are added.
Step 4.Pliers are often useful for loosening the knobs on the FasePrep vortex when removing the lysates.
Step 8.The water bath sonicator develops a film over the membrane used to sonicate the samples.For best results run the sonicator at level 10 for a few seconds to break the film up before adding samples to the water and sonicating at level 5.It may also be a good idea to add some ice to the water bath to keep the samples as cool as possible.
Quality Control: After the extracts are made you can verify efficient shearing of DNA by the following:
1.mix 20μl extract+ 1μl 10% SDS+ 0.5μl proteinaseK
2.incubate 1hr at 37 degrees C followed by 2 hrs at 65 degrees C
3.phenol/chloroform extract the samples
4.chloroform extract the samples
5.EtOH ppt DNA with 10μg/ml glycogen (final concentration)
6.resuspend DNA in 20μl TE
7.treat with 40μg/ml RNAse for 1hr at 37 degrees C
8.run half of each sample on a 1.5% agarose gel with EtBr with an appropriate DNA sizing ladder (sheared DNA should run between 100 bp and 1000 bp)
Step 12.ChIP extracts made as described are generally 15-25 mg/ml