Hi Shang, 

If you have successfully carried out the protocol previously your should troubleshoot each step again.

99.9% times, the white pellet after ethanol precipitation is co-precipitated proteins.

Is there a change in starting material than previous? If yes, then this particular sample may have some proteins which may interfere with protein removal.

Try adding 2% PVP or Beta-mercaptoethanol in your starting buffer along with SDS. This worked for me many times. Protein will definitely interfere with qPCR. If you are low on sample try purifying your isolate with Agencourt beads. They will bind DNA and everything else will be removed.

Gudluck 

Thanks for your answer, Abhinav. The cell samples were collected and fixed in the same way as before. Actually I am now working with the ChIP experiments of a DNA-bound TF and one of its cofactor seperately. The ChIP-qPCR results for the TF were not affected by the pellet conditions while the ChIP-qPCR results for the cofactor were desparately poorer than before. I could now only get 0.02-0.03% input for the enrichment (previous results showed ~0.3%) and the Ct(Cq) values of target gene promoter enrichment and non-related regions showed no significant difference, both approximate 33.

The starting buffer you mentioned is the elution buffer?

Actually I readded proteinase K (Sigma, final conc. 200ug/ml) to the eluted sample and set on 50degreeC heating block for another 1-2h before phenol extraction, but the pellets remained there. The pellets of input samples were always normal.   Could it be the cause of SDS or the glycogen?

Thanks again~

I have used Chelex100 extraction described in the attached paper instead of phenol and it worked very well in several cell lines (however not in yeast for some reason).

Article Fast chromatin immunoprecipitation assay

If you have a lot of nucleic acid (DNA and RNA), you will see white pellets after ethanol precipitation. Nucleic acid pellets are more visible when there is a lot of salt present. The extra salt could cause problems in PCR. You might want to consider doing a second ethanol precipitation. 

Hi. I had similar problem with my ChIP experiments. I added 10M ammonium acetate to the sample while keeping for ethanol precipitation. Add 1/10th of total volume. But do not use ammonium acetate for preparation of DNA for T4 polynucleotide kinase reactions as ammonium ions inhibit the enzyme. Sodium acetate can be used in such cases.

Hi. You might want to try isopropanol precipitation instead, which might even work at RT. It is less prone for salt precipitation. 0,7 volumes might do, but you can go up to 1 volume. It definitely needs one or two 70% ethanol washing steps to get rid of the phenol traces afterwards. A nucleic acid pellet - at least for my eyes - looks brownish instead of white.

Hi Shang,

You asked if SDS or glycogen could be responsible for your observations. First of all, glycogen works as nucleic acids carrier during alcoholic precipitation and a visible pellet will be a good sign. Glycogen is easily soluble in TE-buffer or water afterwards and you seem to have no problems in dissolving it. Not having a pellet after precipitation with glycogen should give you something to worry.

But since you have a low DNA recovery rate and no enrichment of target binding sites in your ChIP qPCR could mean that you have to much residual SDS in your chromatin lysates at the time of Ab-incubation, so the pull down with beads will not work properly. In case you are not doing it already, try to dilute the SDS in your chromatin lysate before adding the Ab and spin it down and remove as much of it as possible.

And as Abhinav said, troubleshoot properly to find out which steps of you protocol might have changed since your last successful experiment.

Hi Shang, did you figure out the problem? I'm running into the same issue for my ChIP re-ChIP with the same antibodies/protocol/cells/buffers that have worked a week before. Even my regular ChIP processed with these samples but phenol extracted/ethanol precipitated the day before worked. I also use proteinase K treatment before phenol extraction. I thought it was too much salt as well since my A260/280 ratio were about 1.33 for both antibody (against a transcription factor) IP and IgG control. I'm currently doing another ethanol precipitation on the same samples but any tips would be appreciated!

Thanks for all the inputs of ideas and suggestions.

Actually I cannot fully confirm where the question were because the recent batches of ChIP went well. 

For the failed trial, I once tried to re-digest with PK and ethanol precipitation and warm the eluted DNA (in ddH2O) on 45C for one hour, the enrichment fold did went up a little.

For Nikki Kong, here are some tips which I think may work in the improvement of reducing the white pellet during ChIP DNA purification:

1.  Add additional Proteinase K to the reverse-crosslink reaction and set in 45C for 1-2h before phenol extraction. Use a new aliquot every time. The remaining PK solution can be used in other experiment such as mice tail digestion. I think the overnight reversal reaction at 65C may sometimes destroy the PK if the temperature of waterbath fluctuated.

2.  Do the ethanol precipitation at RT or shortly than before. Isopropanol suggested by Christian Schröder probably also work but I did not try.  In fact in my recent trials, I shaked the tubes immediately after the ethanol addtion and I can see much tiny string-like turbidities. Then I centrifuge the samples at RT for 10 min. The pellet will be reduced.

3. Do not overdry the DNA and try dissolving the eluted DNA at 45C for 5-10min with gentle shaking. I found this helped in improving the qPCR results.

Hi Alexander,

     For the SDS dilution you suggested, actually I will dilute centrifuged supernatant of sonicated chromatin 10 fold before adding the antibody. Some others suggested in their protocols diluting 5-10 fold.  

   I think you really remind me to think of the SDS in Ab-protein binding. In my previous trials, I found that  the chromatin sample after sonication would show white precipates sometimes. I considered it as SDS and grabbed the tube in my hands for a few seconds to redissolve the white pellets before centrifugation. 

   In your suggestion, would it be better if I let the pellet form and spin them down?

Hi Shang,

thank you for your response. I found the problem: forgot to dilute my samples with TE after over night reverse crosslinking in elution buffer, which contains 1% SDS. I usually do this before RnaseA and proteinase K treatments. This oversight explains why my other samples worked but these two didn't.

Just wondering if you ever figured out exactly where the white precipitant was caming from. I'm having a similar problem, but it doesn't occur ever time. It's also present in my input. I'm doing a phenol/cholorform extraction at the end. At first I thought maybe I was capturing something in the interphase, but even after taking special care, its still there. Any thoughts would be appreciated.

Getting the same white precipitate. Though I have kept it for drying. Has anyone figured out the problem?