I have some problems visualising my sheared chromatin. I seem always to get a long smear from which it is difficult to determine fragement sizes. I am working with primary colorectal cancer cells (SW480). I use a sonicator and have tried using 5', 10', 15' and 30'. I still seem to get this uniform smear. I am wondering if any of you have had a similar experience and have troubleshooted this successfully (images attached).
I purify my DNA after reverse crosslinking a 25ul aliquot. I measure the concentration of DNA on a nanodrop (I question its reliablility for this application). I read usually people load 20-30ng. However I see nothing when I load this much according to the nanodrop. I have to load a nanodrop apparent 5ug to see the smears from the attached file.
Any suggestions will be greatly appreciated.
Best,
Jason
Hello Jason,
From what I see, it seems that your DNA is indeed not sheared enough. I guess from the picture that you load the samples in bromophenol blue. Try using the orange instead, because it migrates quicker and will not mask your DNA. But this will not solve your problem!
Maybe you cross-link too long? Generally 10 min in 1% formaldehyde at room temperature is enough. What sonicator are you using? This will influence your results!
Best,
Germain
I agree with Germain that undershearing could be due to over-cross linking or lack of sonication power. Another possibility might be that the buffer the cells are in is not appropriate for sonication.
With respect to the nanodrop and gel loading, I don't understand why the nanodrop reading is not accurate, if the DNA is actually purified away from potentially interfering substances. (Does the absorbance spectrum look like clean DNA?) It may just be that because there is such a wide range of fragment sizes in your sample that it is too diffuse to see. A single tight band can be visualized at the 10 ng level, but 10 ng divided into thousands of band of different sizes will not be detectable.
Hi Jason,
In addition to over fixation, there could be several reasons for incomplete shearing. Are you isolating nuclei prior to sonication, or going straight into sonication from whole cell lysates? In my experience, sonication will be more successful if you first isolate nuclei and perform a snap freeze on your nuclear pellet before re-suspending in your sonication buffer.
Some other suggestions: Make sure you are quenching your fixation with 0.125M glycine for 5 minutes, and wash your cell pellet a couple times with cold PBS before cell lysis. And as a rule of thumb, try not to exceed 50 million cells/mL in your sonication buffer.
Hi,
We use a sonication buffer with high SDS as this really helps shearing. (50mM Tris-HCl pH8.0, 10mM EDTA, 1% SDS). I use 100ul per 10million cells, aliquot 100-150ul into eppendorfs and use a Bioruptor Pico. 30son 30s off 10 cycles, vortex, then 10 again (depending on cell line).
After sonication dilute 1:10 in dilution buffer (20mM Tris-HCl pH8.0, 1mM EDTA, 0.01% SDS, 150mM NaCl, 1% Triton X-100)
I have used other buffers also with success, I think a key thing is to not include TritonX-100 before sonication as this really reduces shearing.
Hope this helps!
Hi all, thank you for your responses. I am wondering now whether I should purify at all. I spin the nuclei down then lyse the cells in lysis buffer. With these samples I did not treat with RNase. One of my colleagues says that he does not purify and just treats with RNase A then runs the reverse cross-linked sample out on a gel. The reason I think I should not purify is that the Bioruptor manual says that column purification of DNA can be biased towards larger fragment sizes. I have also just made a fresh preparation over the weekend of cells fixed in solution. I was fixing cells whilst they were still adherent the last time. I made some Glycine up and quenched the cells in solution at a final concetration of 0.125M. Adam.
I will measure the DNA whilst still in the lysis buffer before sonication on both the nanodrop and the fluorometer, then shear 10ng/ul as the bioruptor manual suggests in eppendorffs in a volume of 300ul. I think I need to try this first before I start changing the buffers.
Would you guys load around 20ng per well when you're checking chromatin shearing efficiency typically? And also do you use a column purification or do you not purify at all?
Best,
Jason
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