I am trying to standardize chromatin shearing conditions in our lab for doing CHIP.. I am using drosophila wing discs, fix them with 1% formaldehyde for 10 minutes, lyse them and then shear them using bioruptor. my problem is that I am not able to get reproducible results. Moreover, it seems that shearing is not complete because most of the times I am getting DNA even above 800bp... sometimes about 1.2kb. DNA smear is from 200 to almost 1 kb.
Thanku so much :) but my problem is not what sonication time and cycles to use. my problem is reproduciblity of results. so i think there is problem in processing of cells upto sonication. with cell types that i am using, i have got 100-300 bp when i use 30/30 15 cycles. but problem in reproduciblity. Size range that i am interested in is 200-500bp.
Hi,
I wouldn't recommend any specific time concerning your sonication problem, since each cell line from any organism and each machine will behave differently. In addition cross-linking time and reagent is influencing the sonication efficiency. I would recommend to test a range of different times for your sonication without changing cross linking time, volume and temperature used in your sonication. Madhusudhan suggestion of the starting point is pretty good. We are using a Biorupter for sonicating yeast cells at 4°C with a program containing 3 cycles of 30 sec on, 30 sec off each 10 min. In between of the cycles the samples are chilled on ice for 2 min and the water is changed. Newer machines will use a continuously ice/ethanol bath where no interruption is required.
I also work on drosophila wing discs. The protocol we use is fixation FA 1,8%, and 15 cycles of 30 secON 30 sec OFF High power on a diagenode bioruptor. Sometimes, with no explainable reason, one tube on the six sonnicated in the same time have foam on top of the liquid and this impairs the quality of shearing. We also seen variability in the results when too few material were used (below 50 wing discs for one IP Q-PCR) . How many discs do you dissect for each sonication ? If you want, you can find our protocol on my last publication. Hope you'll get with it.
Thank u Thomas.
Thank you Kenneth!
Anne, i would definitely like to see your protocol. i also use diagenode bioruptor. Moreover i am also planing to do qPCR after immunoprecipitation. i am sure that it will help. Regarding dissection of discs....i am very confused about how many discs to take. I am doing it for tumorous wing disc. considering that 1 tumorous wing disc contains more than 200,000 cells, no of discs that i would need will be less...right?
i have tried with 35, 45, 55, and 70 wing discs, giving 10 minutes fixation time and 12 cycles of 30 on 30 off. (6 minutes sonication).
but as i told previously I am not able to get completely sheared chromatin. so cant really distinguish between them. and yes I am using 1.5 ml eppendorf tubes.
Make sure you lyse and sonicate your disks in 1% SDS Lysis Buffer.
Concerning your second problem of "insufficient" break-down of x-linked gDNA: in my experience sonication efficiency in a bioruptor (even with the "high" settings as recommended) is not that great, especially in standard Eppendorf cups. Do you add (acid-washed) glass beads to your samples (definitely improves shearing efficiency)? For tough material I'd definitely recommend a tip sonicator if available. Yet, on such machines you can handle only one sample at a time which is, of course, not really advantageous if you are going for great reproducibility... By the way: what is your factor you try to IP? If it is part of a multisubunit complex (e.g. chromatin remodelers et al.) and is only indirectly tethered to the DNA you might think twice before applying too harsh sonication conditions, since you'll start to shear/destroy such multiprotein assemblages and run the risk to lose your protein of interest before starting the critical IP step.
Good luck!
Anyone suggests me....I am doing ChIP from lipid containing seed. The problem is, when I check the fragmented DNA in 1.5% agarose gel after sonication, I got around 500bp band not like smear. After RNase A and Proteinase K treatment and Phenol: Chloroform purification, I found no band on the gel. I am using JY 92-IIN sonicator with 90% power, 20sec on, 60 sec off and 8-10 cycle. Can anyone suggest me....
You can try calorimetry as suggested by NIST
"Taurozzi, J. S., V. A. Hackley, and M. R. Wiesner. "Preparation of nanoparticle dispersions from powdered material using ultrasonic disruption." NIST special publication 1200.2 (2012)."
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