7 replies to this topic

[TracyDuke]

I am currently attempting to perform a ChIP sequencing following the Myer's Lab ChIPSeq protocol. I am having a difficult time to shear DNA to the desired size (<600 bp). I am using a 130 Watt Ultrasonic Processor. According to the protocol, cells were lysed in Farnham buffer then sonicated in RIPA. I used up to 8 X 30 seconds pulses, 50 seconds between each two pulses. But still the chromatin DNA was not sheared (See attachment). Could anybody give me some advices or comments. I'll greatly appreciate that!

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[pcrman]

Hi,

From what shown in the picture, the sonication is not enough, although you have used relative high amount of pulses. What is the sonication volume and did you get foaming during sonication?

[KPDE]

TracyDuke, on Jun 5 2009, 07:00 AM, said:

I am currently attempting to perform a ChIP sequencing following the Myer's Lab ChIPSeq protocol. I am having a difficult time to shear DNA to the desired size (<600 bp). I am using a 130 Watt Ultrasonic Processor. According to the protocol, cells were lysed in Farnham buffer then sonicated in RIPA. I used up to 8 X 30 seconds pulses, 50 seconds between each two pulses. But still the chromatin DNA was not sheared (See attachment). Could anybody give me some advices or comments. I'll greatly appreciate that!

One problem I had early on with sonication was that I was trying to sonicate too large of a cell pellet. Increasing viscosity decreases sonication efficeincy so you might try resuspending your pellet in a larger volume (even if that means splitting it in half and sonicating both halves separately).

[TracyDuke]

Thank you very much for your replies!

I did not get foaming during sonication. 35M cells were lysed in 1ml lysis buffer and split into 2 1.5 eppendorf tubes. Sonication was done in 500 ul volume.

I compared lysing the cells in 1% SDS ChIP lysing buffer and in RIPA, which has 0.1% SDS. Under the same sonication condition, the DNA was sheared much bettern in 1%SDS than in 0.1% SDS. I am wondering if I eventually use 1% SDS, how much shall I dilute the lysate before for the IP step. Some protocols suggest 1:10, but this will substantially increase the volume and much more antibody amount will be needed. Some protocols suggest 1:2. Does IP work with 0.5% SDS? Does high SDS denature antibody?

[KPDE]

TracyDuke, on Jun 10 2009, 06:20 AM, said:

Thank you very much for your replies!

I did not get foaming during sonication. 35M cells were lysed in 1ml lysis buffer and split into 2 1.5 eppendorf tubes. Sonication was done in 500 ul volume.

I compared lysing the cells in 1% SDS ChIP lysing buffer and in RIPA, which has 0.1% SDS. Under the same sonication condition, the DNA was sheared much bettern in 1%SDS than in 0.1% SDS. I am wondering if I eventually use 1% SDS, how much shall I dilute the lysate before for the IP step. Some protocols suggest 1:10, but this will substantially increase the volume and much more antibody amount will be needed. Some protocols suggest 1:2. Does IP work with 0.5% SDS? Does high SDS denature antibody?

I've always read that greater than 0.2% SDS is problematic for antibodies in IP but I've never actually done an optimization with different amounts of SDS.

[timjim]

I think 1% of SDS is good enough to lyse the cells. I do agree with the viscosity of the cells suspension. Now I did 2 and 4 times dilution of the cells and longer sonication to see the best condition for it.

Just a stupid question, dont you think if we use protease inhibitor, there will be more cell protein present in the samples and thus that might actually inhibit the sonication? Maybe it is too far fetched. =)

[hamidk]

Hi Tracy,

Shearing chromatin by using sonication has some inherent issues which has been shown and discussed recently:
1. probe and bath sonicator generate a tremendous amount of heat this can not only cause denatureation of the proteins, but also cause thermal biased shearing of DNA. There has been some indication that the high and difficult to control temperature fluctuations when using a bath and probe sonicators can cause acetylation of histones and associated proteins.
2. the long 10-15cm wavelengths generated by bath and probe sonicators are very difficult to control. They lack focus and slight changes in buffer compositions, cell number, and cell types can affect reproducibility.
3. Cross contamination and time consuming clean up steps are continued concerns for probe based sonicators.

Covaris recently released two tested protocols for chromatin shearing using the Covaris AFA technology. the protoclols are http://www.covarisin...f/pn_400066.pdf and http://www.covarisin...f/pn_400067.pdf

I hope this information is helpful to you.

Thank you

Hamid

[rqliang]

I always get around 150 bp small fragment. But I want 200-1000 bp fragment. Are you using VibraCell VCX 130? How much is the amplitude?