For ChIP-Seq analysis I've seen people describing water-bath sonicators, probe-sonicator, sonicators in the Hz range, in the kHz range...different W...
Can anyone help in explaining which (and why) are the key parameters or specificities I should focus on to choose a sonicator optimal for ChIP-Seq?
Thanks!
Hello Santos, i have used the probe sonicator and the Bioruptor (the one with a waterbath) for Chip-seq. The main determinant is really what you have available. However, the Bioruptor has the advantage of keeping your samples cool (around 4 degrees) because the samples will heat up pretty fast while with the probe you have to try to keep it in on ice while sonicating or what is more common is to have breaks in between pulses to keep on ice.
Whichever method you use, you would still need to run a trial with your samples to figure out how many pulses/cycles will be optimal. So something like 5, 10, 15, 20 cycles... then run on a gel to determine what number of cycles gives you fragments at the size you want (usually 200-500bp).
lol...that's okay.
Okay, there are two kinds of sonicators (i think)... the probe and the waterbath (Biroruptor) but both have a range of amplitudes to select from.
My lab has a probe sonicator with Voltage=120/220Volts, 50-60Hz and a freq. of 22.5 KHz. This is not adjustable (i think). amplitude however is. You have to run several sonication trials to find what works best. i had to run alot! i used a low, medium and high value at 5, 10, 15 and 20 cycles. The medium worked out better. The high was good too but it was too intense for my samples. Then i tried again with just the medium and the same number of cycles just to check for consistency. Then i ran another trial with the one cycle that seemed to work best (15) but with several samples to again check consistency.
The Bioruptor i used belongs to another lab in my Faculty and i don't recall its exact features at the moment. But you could set it at low, medium or high intensity. Set the number of cycles...how many seconds ON, how many seconds OFF. i used High intensity with this.
If you are looking to buy a sonicator, then you can look around and compare. But if you are trying to figure out if an available sonicator is suitable, my guess is anyone would work, you just have to optimize it for your samples. The Hz/Khz is not the determinant.
This might help:
For the probe
http://www.sonicator.com/literature/manuals/Catalog.pdf
An excerpt
The ultrasonic frequency is fixed at 20 kHz and does not fluctuate during sonication.
The Sonicator continuously calibrates itself to ensure consistent output. Processing
time and amplitude (intensity) are adjustable.
For the Bioruptor:
https://www.diagenode.com/categories/bioruptor-shearing-device
i hope this helps
Dear
-Direct Probe Sonicators use a probe immersed directly into a sample tube. They deliver a whole lot of power to the sample, which makes for short sonication times. That said, this isn’t always ideal for ChIP applications.
-I recommand you Cup Horn sonicators which use a separate water bath to indirectly deliver energy to your sample. Since this type of a water bath approach is less intense than direct sonication, the duration of sonication required can be a bit longer. Frequence of 30-40 KHz is need
For more information check this link: http://epigenie.com/epigenie-guide-chromatin-shearing/
Thank
Marius
Bioruptor is so far the best option nowadays.
In any case, based on published reports yuo woould need to find tteh best seetings for your samples experimentally, as the extraction buffer, lysys solution , type of plastic tubes, etc ... influence sonication.
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