Hi Joshua,

I found this very nice image for sonication online (link below)

https://www.activemotif.com/images/products/chip_sonic_gel.png

You should aim to get a DNA smear between 200-600 bp for most ChIP protocol. It will be more helpful if you provide more information about your sonication attempts (volume, pulse, output voltage...etc). I tend to do 12-15 cycles  of 10 sec on/off pulse at max voltage for smaller volumes (200-500 ul) in eppendrof tubes.

Good luck

hi,

You can find nice pictures of sheared chromatin at https://www.diagenode.com/files/protocols/bioruptor-pico-chromatin-preparation-guide.pdf

Thanks for your helpful responses! I was hoping to see a couple examples of gels that people have actually run and proceeded to take through sequencing or qpcr with positive results. Also I'm looking for some clarification since I've read many protocols and read some people say anything below 1000 bp while others restrict to about 600 bp saying that the avg bp length should be around 200 bp. Does this enhance sequencing results?

Here is a gel I ran recently. I did 10 cycles of [15s on 45s off at 50% amplitude], which reads out to about 150-160W when sonicating. This is pretty clearly undersonicated. I'll post my next attempt right after this because it doesn't seem like I can post 2 images.

So a few days after this attempt I tried a variety of other programs. I used about the same amount of input material for all runs on this gel. For each program I tested there is a treated and untreated sample right next to each other.

At first I tried increasing the amplitude only, which appears to have minimal effect on the end result, although it looks more sonicated than my previous gel. While increasing the number of cycles seems to have more effect. I would lean towards using the 15 cycle 50% amplitude samples for an IP however it still looks like it could use a little more sonication. Any tips on this? I would want to try to avoid oversonication, so perhaps increasing the number of cycles and reducing overall amplitude slightly?

Hi,

Did you perform de-crosslinking? it looks like cross-linked matireal ( a distinct sharp band in the middle of the gel is typical for cross-linked chromatin).

For accurate size determination of the chromatin fragments, the reversing of the crosslinks, including RNase treatment followed by DNA purification, is advised. The estimation of the size of chromatin fragments without the reversion of cross-linking is not precise. The presence of the crosslinks retards electrophoretic migration resulting in a misinterpretation of fragment size

See an example in the attachment, from  the same chromatin

Yes I did uncrosslink for ~10 hours with NaCl, followed by treatment with RNase and proteinase although I did not purify the DNA. I can purify the DNA next time and run again. Would the unpurified DNA give an inaccurate size representation?

Yes, you should purify DNA, all unpurities may retard the migration in a gel.

What type of tubes are you using and what is your sample volume? 

Rajaei - It looks like that gel may be overloaded? According to this forum http://seqanswers.com/forums/showthread.php?t=9585 this may make sonicated DNA appear to be more sonicated than it actually is, which is a problem I have had when getting CHIP'd DNA back. When you have a gel that looks like that which you posted, did you ever run a tapestation of the DNA you chip'd to see what the size range of DNA fragments was?

Irina - I took another aliquot of chromatin, uncrosslinked w/ NaCl at 65 O/N and purified w/ phenol chloroform and ethanol precipitation. I ran the attached 2% agarose gel visualized w/ SYBR safe at 100V for 50 minutes. I think it definitely looks more clear purified, with the 15 cycles 50% amplitude still giving the best result. Although I now have concerns that I am overloading the gel, see this forum: http://seqanswers.com/forums/showthread.php?t=9585 ...do you have any advice on this? I spec'd the DNA and it appears I am loading between 2-5 ug of purfied dna in a lane. According to the forum overloaded gels can make DNA appear to be more sonicated than it actually is. I plan to dilute this and run again, but I have run some other samples a different concentrations and around 1-2 ug appear to give better results, though I would still like to confirm on a tapestation, so I will update the this forum with my findings.

Marc - I am using polystyrene tubes from evergreen scientific (214-3721-010). In in this experiment I am using 200 ug per tube of chromatin in a 300 ul volume of lysis buffer. I adjust the volume of h20 in the cup horn prior to sonication to the level of the samples in the tubes and cool the h20 with a small amount of ice around the outside edge of the cuphorn. Should I adjust the volume or use different tubes? Would you have a different suggestion on how much chromatin to sonicate at one time?

In terms of set up, sounds like you are fine.  Its possible that you need to run more cycles.  If that doesn't give you the desired fragment size you can increase the amplitude to 60% and run another time course trial.  Feel free to call me anytime.

Hi,

For accurate fragmentation assessment, the sheared chromatin should be analyzed on a 1.2 - 1.8% agarose gel. The optimal DNA amount from sheared chromatin is around 300 ng per lane. A serial dilution from 100 ng to 500 ng could be run. Do not overload the gel as the migration of large quantities of chromatin on an agarose gel can lead to poor quality pictures which do not reflect the real DNA fragmentation. The minimum amount of sheared chromatin that can be visualized in an agarose gel corresponds to 60,000 cells equivalent.

Both the pre- and post-staining of the agarose gel with ethidium bromide or SybrSafe dye can be used for visualization of sheared fragments. Some slight differences might be observed between post- and pre-stained gels. Post-staining eliminates any possibility that the dye interferes with the migration and ensures an even background noise. However, pictures are usually less clear and bright with some background noise. If pre-stained agarose gels are used, it is advised that the electrophoresis buffer contains the stain in the same concentration as in the gel. If the stain is present in the gel but not in the buffer, the gel will result in uneven staining because the free dye migrates towards the top of the gel leaving the bottom part with no stain. Therefore, the background noise becomes non-uniform.