High background with biorupter - (Mar/08/2010 )

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?

-annabellak-

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

-jonny_boy-

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.

-annabellak-

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.

-KPDE-

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?

-NemaToStella-

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.

-Dukey-

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.

-annabellak-

annabellak on Mar 19 2010, 12:14 PM said:

Glad you figured it out.

-KPDE-