yanks1ny, on 16 June 2012 - 08:06 AM, said:
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.
Thanks a lot for your respond.
I'll try to use TPX tubes and remove all the ice in later sonication. SDS precipitation maybe a serious problem. During sonicating, the precipitation at the bottom would affect the ultrasound from the tank bottom, wouldn't it? Gently warming up the tube may be a choice. How did others deal with the problem?