ChIP assay - Foaming in sonication and other woes - (Jan/27/2005 )
Micrococcal nuclease has also been used to fragment the chromatin.
Hi, I've been trying to ChIP for the first time and I'm having trouble sonicating so i just joined this forum
To avoid heating, hold the tube in ice water during sonication.
Does this mean that the probe should touch the bottom of the tube? I've been using this trick to avoid foaming in sonicating proteins but it seems that cells fixed in formaldehyde become somewhat more resistant to sonication, so i didn't get any sheared chromatin at all sonicating this way.
I used Sonics Vibracell VX400, 30% power with 5s on/7.5s off pulse (this sonicator doesn't support pulses longer than 10s ) with total of 20/40/60 cycles. Help.
You don't have to touch the tip to the bottom of the tube, just make sure it is sufficiently submerged, I use 500ul for sonication, a deviation from the upstate kit I use, but allows for sonication without foaming, and gives more efficient sonication in my hands. Don't go too high, misonix argues that with volumes over 1 mL it is very difficult to shear the chromatin.
Sonix82- what setting do you use and how long (how many pulses etc.) do you use? We recently purchased a Fisher 500 and cup horn, but it doesn't seem to be shearing the chromatin.
Hello fellow ChIP'ers... I have also been having sonication problems recently and I was hoping for some advice.
I have been doing ChIPs for a number of years and recently had to change machines. While trying to optimize my new sonicator, I have been noticing that under all conditions I try the DNA just won't shear very well! My average fragment size is around 4kb... which is way too big for the promoter scanning studies I would like to try. I have tried dropping the Formadehyde conc. from 1% to 0.5%, I have tried different output settings and total times and nothing has worked. I have even tried the cup horn mentioned in this forum, but that seems to be even worse in my hands than direct probe. I am completely lost!
Here are my questions: (1) Do you think the sonication vessel makes a difference? I do my sonications in 15ml Falcon tubes containing 1ml of lysate... I just feel I have the best manual control of the tube that way, but I was wondering if that could be part of the problem (i.e.- interfering with sonication of the sample).
(2) Once a sample foams, is it impossible to ever get a good shearing pattern from the sample? When I have had foaming, I stop my sonication immediately and allow the sample to "settle" for a few minutes before resuming, but maybe this is not the proper approach.
(3) Has anyone ever had variable results with different lots of formaldehyde?
(4) Should one see a change in the turbidity of the lysate after sonication (ie- should it appear more clear)? I see that many of you had said that 500ul lysate plus lower sonication power does the job. I will try that immediately, however in my hands a lower power level does not "clear" the solution as well. Any thoughts?
Sorry for the long post, guys. I am trying to graduate and very desparate for some help!!!
-Appreciating your help in advance!
Hi I had the same problem 2 yrs ago. hopefully you solved it by now. 1.5 ml conical tube is the best option. Mostly I used to have the problem when I used to hold the tube in hand. If you somehow mechanized it this problem evaporates! I use a home made version (a plastic container filled with ice and water and I drilled a hole in the cap that stabilizes your eppendorf tube in place an easy version.) and as you are using branson 250 you could automate the run with your pulse pause. dont have to look in to it at all until its done! also make sure that the probe is almost at the bottom when you are setting it up! but not touching the tube.
Have you noticed any difference in sonication efficiency when using different detergents in the sonication buffer? (for example Tween vs. NP-40 vs. SDS?) I wonder if that could be related to the foaming issue....
Also, which type of vessel do you use for sonication? I use 2 ml eppendorf tubes, but the instruction manual of my sonicator recomends metal or glass vessels, better than plastic ones, because of heating considerations.
And last but not least, have you noticed differences in the sonication quality when using different cell lines? For some reason, I get really small fragments with 3T3 cells but not with 10T/2, and both of them are fibroblast cell lines. I don't change the conditions and sometimes I perform the experiments in parallel
Any thoughts or suggestions will be very welcome. Thanx!
The answer to your problem may be that you are using a probe sonicator, which doesn't give you the chance to sonicate in closed tubes.
This mail should really draw all your attention :
Diagenode is proposing a truly innovative system of sonication called Bioruptor which is perfectly adapted for a number of applications in molecular and cellular biology where an optimal reproducibility is of major concern.
With the Bioruptor, it is now possible to sonicate samples in sealed tubes (Eppendorf or Falcon 15 or 50ml), without introducing a probe into the sample as traditional sonication methods require. This enables the avoidance of tedious manipulations but also the prevention of any contamination between the samples.
The Bioruptor is perfectly adapted for the ChIP assay where optimal fractionation of DNA is required. Up to 12 tubes can be sonicated in parallel and 200 to 500 bp DNA fragments are obtained in minutes (minimal sonication volume: 5 microliters).
The system is widely used in key reference labs for ChIP assay in Japan, Europe and now in North America.
We would be very pleased to propose you a two-week trial of the system in your laboratory, in order for you to test the Bioruptor perfectly.
More information can be accessed via our web page at the following address:
Looking forward to hearing from you soon.
Someone always told me there is not dumb questions, but this might be one. Im starting my ChIP experiment by optimizing the DNA shearing by sonication. However, after 4h at 65 degrees with NaCl (upstate protocol) I did phenol chlorophorm extraction and resuspended the DNA in TE buffer. I ran the gel and had a larg smear (4kb to 200nt) so i assumed the sonication did not break up the RNA. So i treated the sample with RNase A, and then was left with a single band at around 4kb or so.
So, my question is, should I treat the samples with RNase A after phenol chlorophorme extraction.
Another question, im assuming the power setting in amplitude is in percentage (30%), is there another power setting I should worry about.
Finally, is an eppy tube an epindorph tube. Thanks alot for any info.
There are no dumb questions just jerks who act like there are!!
an eppy is technically a 1.5mL eppendorf tube but 1.5mL tubes from any supplier should be equivalent
30% power should be okay I use the "maximum microtip level" on my misonix sonciator...
I had similar problems before, here is a previous post....
Here are some suggestions so that others hopefully won't have the same problems as I had.
(after reversing crosslinks and phenol/chloroform extraction and precipitation) You should add RNase to the resuspension buffer to ensure only DNA shows up on the gel (this is a good reason to precipitate because although the RNase does have activity in the lysis buffer, it is not sufficient to completely degrade the RNA) And make sure that you check sonication on a sufficient amount of DNA, i can see about 850ng. This is equivalent to 25ul of lysate when 10^6 cells are lysed in 200ul (Upstate protocol)
The 850ng estimate is based upon the celera prediction of 6.6pg of genomic DNA/cell and assumes 100% lysis, extraction/precipitation, etc... so if you are quantitating directly, 500ng DNA is probably enough to see on the gel.