ChIP: SDS precipitates during sonication...any ideas? - (May/19/2008 )
I am using 1% SDS for my sonication and I've noticed that the SDS frequently comes out of solution during one of the early ice bath incubations. I've also noticed that when this happens, I seem to lose substantial amounts of my DNA.
First, does anyone have an explanation for why SDS precipitation may correlate with loss of DNA? Second, how do I avoid the precipitation to begin with? I've thought about warming the tube slightly between my fingers between pulses, but this would seem to defeat the purpose of the ice bath incubation.
Thanks!
First, does anyone have an explanation for why SDS precipitation may correlate with loss of DNA? Second, how do I avoid the precipitation to begin with? I've thought about warming the tube slightly between my fingers between pulses, but this would seem to defeat the purpose of the ice bath incubation.
Thanks!
Sonication generates sufficient energy (and heat), so as to keep SDS in solution. If you see SDS ppt, that means your sonication is not set at ideal settings. Increase the power (if 25%->50%->75%) depending upon the kind of sonicator you are using. Do not use body heat :-) Obviously, you still need to keep the samples dipped in ice-water.
If you use water-bath type sonicator, make sure your tubes are dipped in water, because soncation waves are generated and work only in the water, not if tubes are hanging in the air. But as the water-bath may be in the cold-room, your SDS would ppt in absence of sonication heat.
I'll try increasing the power, but would sonicating in the presence of SDS precipitate damage the DNA?
I observe the following. After homogenization, I add SDS to 1%. I then take a 10ul sample and put it on ice for later analysis. I sonicated the remaining chromatin, taking 10ul samples periodically and putting them on ice. I then perform the de-crosslinking overnight, followed by RNase and Prot K treatment according to the Upstate EZ-ChIP kit instructions. I finish by purifying the DNA using the columns in the kit. When I run my samples out on a gel, I see a bright high molecular weight smear in the first sample and diminishing amounts of DNA in each subsequent sample. It rapidly gets to the point where I can barely see the DNA on the gel.
Then you should be just fine.
May be you are using less number of cells (2X10^7 as per kit inst), so that you don't get sufficient amount of DNA to be very visible on the gel. You need to use as much DNA as it takes for you to confirm that all sonicated DNA is between 200bp and 1-2Kb area.
Presence of some SDS crystals in the beginning (prior to, and in the initial sonication rounds) does not damage the chromatin. Just make sure that they do disappear by the end of sonication. The bottom line is that your sonication conditions should be adjusted to ultimately get .2-2 Kb fragments.
Then you should be just fine.
I am taking 10ul of sample to run on the gel at each point and I can see tons of DNA after the homogenization and prior to the first sonication. But when I take a sample after each sonication pulse, the amount of DNA is diminishing with each pulse.
Can you elaborate on this? Why is it okay to have SDS crystals early in the process, but important to get rid of them by the end?
That is because the DNA is being spread out. anything more than 20Kb gives you a single band and hence bright, whereas something spread out between .2 and 2 diminishes etbr fluorescence per unit area, and so when it goes below 20ng at any point, it does not show up well.
Oh, that is nothing. That just shows you that sonication was probably working, becasue heat was generated sufficient to melt SDS crystals. I may be wrong, but I don't think spinning down the crystals at the end of sonication would also bring down chromatin fragments. They would remain in solution no matter what. But bringing down the crystals would decrease the SDS effective concentration for the next step, which has been optimized keeping in mind 1% SDS conc.
Have you ever considered that your sonication maybe to strong?
If you loose DNA after every pulse, it is no good news..
Is losing DNA a symptom of sonication being too strong? I would have thought it would just result in a low molecular weight smear. Is it possible to shear it to the point that it won't stick to the column?