yanks1ny

Hey Jancho,

Unfortunately, we were having a very similar problem with the BioRuptor. Sometimes, it would work very well (i.e. <500 bp fragments) and then other times, it wouldn't work at all (i.e. huge smearing). We tried many different solutions, but in the end, wound up having to use another lab's Covaris E210 machine.

That being said, maybe some of the things that we tried could be helpful to you, given that we used a different cell line:

-For our sonications, we tried using different buffers--1% SDS (as you are doing), 0.1% SDS, and a buffer without SDS (with sodium deoxycholate and N-lauroyl sarcosine). When using the 1% SDS buffer, we limited the amount of time the samples were on ice. We would add the buffer then go to the BioRuptor without putting the samples on ice. In addition, a friend had suggested gently warming up the tube between each sonication step (by rubbing the sides of the tube) to try to decrease the chance of SDS precipitation.

-I agree about the problem with the ice decreasing the sonication efficiency. In fact, although I'm not positive, it's even possible that slight differences in the amount of ice (however little) could contribute to the variability that you are observing. Our BioRuptor is in a cold room, so that decreases the need for a large amount of ice. Right before using the BioRuptor, we added ice and mixed it until it melted. The water bath usually hovered around 2-4ºC. We tried not adding any more ice to the machine and, after 5 minutes at low power, we tested the temperature and it never really got above 10-11ºC. I know that the manual says that the temperature should not exceed 10ºC, but it's at least worth trying out to see so that you don't accidently add different amounts of ice.

-I'm sure that almost everyone that will respond to this post will suggest using the more thin-walled tubes for sonicating, specifically the TPX tubes as opposed to Eppendorf tubes. From our experience, when the sonication worked, the shearing looked MUCH better using the TPX tubes vs. normal Eppendorf tubes. Again, we could not always reproduce this....

We tried to deal with our BioRuptor problem for about 2-2.5 months, but we simply were unable to get consistent shearing. I sincerely hope that you are able to solve your problem.

KPDE: I had seen that suggestion (about the cooling, circulating water system) in some of your previous posts and, in theory, we could do it although the BioRuptor isn't exactly ours but rather it belongs to our department. That being said, I still find it strange that it works for other people in different labs, but has been inconsistent for us.

Update: So, we used the Covaris once again with the same water level as the first time and it seems to have worked, albeit not as well as the first time around (but still, all in all, the sonication was decent). We did a time point and tested both sh infected and non-infected cross-linked HeLa cells and, surprisingly, there was a slight difference--there was definitely an observed a delay when the cells were infected (with just shLuc), so it does seem like simply infecting cells can have an effect on sonication times. Unfortunately, the other member of our lab was not able to see good sonication, BUT we think it's because she didn't properly reverse cross-link her samples. If we can re-produce this again, I think we should be okay.

That being said, I'm still not sure why the BioRuptor isn't consistent for us....

chabraha: in our actual experiment we have both a shLuc control or an shRNA against our gene of interest. Unfortunately we no longer see good sonication with either samples.

Memari: you're right, originally we were using normal eppendorf tubes, but then we switched over to the 1.5 mL TPX tubes. As I had mentioned, during the test run with the untreated cross-linked cells, the sonication looked beautiful with those tubes. However, once we tried again with our shRNA treated cross-linked cells, we no longer saw good sonication.