sonication problems! - (May/25/2009 )
Hi, all, I am sorry but I have to come here for help again. I have two questions on sonication.
One is about my sonicator, Vibra-Cell 130. I think it is a very old model since it only has two regulatory knobs, one for out power and another for working type. Does anyone use the same machine? My question is when I do sonicarion which working type I should choose, pulsed or continuous? Is there much difference between these two types?
Another is about my operation. When I do sonication I put samples on ice all the time but I often found there were some floccule pellet forming in cell lysate. I guess that is SDS separating out. My question is at this moment if I should continue sonication regardless of the pellet or I should warm the lysate to thaw the pellet prior to sonication? Maybe that is a simple question for expert but really confusing me a lot.
Many thanks in advance for any kind help!
Ge
yg_eagle on May 25 2009, 05:32 PM said:
One is about my sonicator, Vibra-Cell 130. I think it is a very old model since it only has two regulatory knobs, one for out power and another for working type. Does anyone use the same machine? My question is when I do sonicarion which working type I should choose, pulsed or continuous? Is there much difference between these two types?
Another is about my operation. When I do sonication I put samples on ice all the time but I often found there were some floccule pellet forming in cell lysate. I guess that is SDS separating out. My question is at this moment if I should continue sonication regardless of the pellet or I should warm the lysate to thaw the pellet prior to sonication? Maybe that is a simple question for expert but really confusing me a lot.
Many thanks in advance for any kind help!
Ge
For sonication with a probe I prefer pulsed sonication. I would do the same amount of time, just broken up into pulses.
As for SDS coming out of solution, it seems like you shouldn't be using so much that it could come out of solution. I know that in my own case, I get white "precipitate" but it's lipid which isn't solublized, since it floats.
I agree with KPDE, you should use pulse mode. There must be a short interval (10") between pulses to allow the lysate to cool down. I prefer sitting the tube in iced water instead of "dry" ice. This will ensure constant cooling of samples.
Hi, KPDE and pcrman, thank you so much for the response and so sorry for the late reply.
As for the SDS coming-out issue, I use the normal concentration of SDS in lysis buffer, 1%. KPDE, do you mean it is a little high? In fact, I have used the method pcrman recommended that I put the lysate in wet ice rather than 'dry' ice during the sonication. I am just not sure if it is all right. Thanks for pcrman's helpful confirmation.
BTW, pcrman, is the 10 sec interval too short? Many protocols say the interval should be no less than 30 sec. Of course, for me, I hope the interval time as soon as better. You guys don't know my sonicator is not an automatic one. I have to choose pulsed option, turn on output power knob to 30% option, then turn off it myself in 15 seconds. Since I had many samples, I was often frozen in the cold room.