We've just switched from a probe sonicator to a biorupter. Since changing, I've been having problems with high background in my no antibody control samples and negative PCR controls. I'm wondering if its a solubility issue. Has anyone else had similar problems?
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#2
jonny_boy
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Posted 09 March 2010 - 06:15 AM
Hey Annabella,
Not sure if this response will help but here we go anyway! Did you re-optimise your sonications for use on the biorupter? If not that is probably the best place to start. Also, did you check your shearing on an agarose gel?
The reason I ask is that in my experience the biorupter is not as efficient as a probe based sonicator and usually requires a higher number of pulses. If you simply switched your entire protocol to the biorupter without checking shearing your background may well be caused by insufficient shearing (greater than 2-3 nucleosomes present).
Hope that helps a little!
Jon
Not sure if this response will help but here we go anyway! Did you re-optimise your sonications for use on the biorupter? If not that is probably the best place to start. Also, did you check your shearing on an agarose gel?
The reason I ask is that in my experience the biorupter is not as efficient as a probe based sonicator and usually requires a higher number of pulses. If you simply switched your entire protocol to the biorupter without checking shearing your background may well be caused by insufficient shearing (greater than 2-3 nucleosomes present).
Hope that helps a little!
Jon
#3
annabellak
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Posted 09 March 2010 - 06:34 AM
Thanks for the reply Jon. We have checked the shearing, and it looks great - around 300bp. Perhaps there is still a small amount of high molecular weight DNA that I can't see on the gel.
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KPDE
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Posted 11 March 2010 - 01:39 PM
annabellak, on Mar 9 2010, 06:34 AM, said:
Thanks for the reply Jon. We have checked the shearing, and it looks great - around 300bp. Perhaps there is still a small amount of high molecular weight DNA that I can't see on the gel.
Have you noticed any change, after switching to the Bioruptor, in the size of the residual pellet when centrifuging after sonication.
#5
NemaToStella
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Posted 16 March 2010 - 09:56 PM
Hello annabella,
this is just a wild guess as I can't see a good reason why you would experience a higher background. I've noticed though that the insoluble chromatin pellet after sonication in the bioruptor seems to be looser/more easily perturbed compared the a "tip sonicator pellet". Could it be possible that you aspire some of the pellet which then sticks to your beads?
this is just a wild guess as I can't see a good reason why you would experience a higher background. I've noticed though that the insoluble chromatin pellet after sonication in the bioruptor seems to be looser/more easily perturbed compared the a "tip sonicator pellet". Could it be possible that you aspire some of the pellet which then sticks to your beads?
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Dukey
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Posted 17 March 2010 - 03:01 PM
annabellak, on Mar 9 2010, 06:34 AM, said:
Thanks for the reply Jon. We have checked the shearing, and it looks great - around 300bp. Perhaps there is still a small amount of high molecular weight DNA that I can't see on the gel.
Be careful, if you get any lower than 300 bp, you could be looking at RNA.
#7
annabellak
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Posted 19 March 2010 - 12:14 PM
Thanks for your suggestions. Yes - the pellet is a little larger after switching to the Biorupter, and I think you're right NemaToStella, the pellet is a little looser.
The samples are well RNase treated, so I'm hopeful that the sonication is real and not RNA.
I think the background was due to insoluble material as I managed to get rid if it by adding an extra spin after I diluted the chromatin - I actually saw a small white pellet after the second spin. I'm now back to around 100 fold enrichment over my PCR negative control.
The samples are well RNase treated, so I'm hopeful that the sonication is real and not RNA.
I think the background was due to insoluble material as I managed to get rid if it by adding an extra spin after I diluted the chromatin - I actually saw a small white pellet after the second spin. I'm now back to around 100 fold enrichment over my PCR negative control.
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Posted 20 March 2010 - 10:01 AM
annabellak, on Mar 19 2010, 12:14 PM, said:
Thanks for your suggestions. Yes - the pellet is a little larger after switching to the Biorupter, and I think you're right NemaToStella, the pellet is a little looser.
The samples are well RNase treated, so I'm hopeful that the sonication is real and not RNA.
I think the background was due to insoluble material as I managed to get rid if it by adding an extra spin after I diluted the chromatin - I actually saw a small white pellet after the second spin. I'm now back to around 100 fold enrichment over my PCR negative control.
The samples are well RNase treated, so I'm hopeful that the sonication is real and not RNA.
I think the background was due to insoluble material as I managed to get rid if it by adding an extra spin after I diluted the chromatin - I actually saw a small white pellet after the second spin. I'm now back to around 100 fold enrichment over my PCR negative control.
Glad you figured it out.
