ChIP sonication problems...can I use 20 sec pulse? - (Jul/17/2006 )
Hi,
I'm having trouble sonicating-tried 1.5 power, 40% output, 10 pulses 8 times with 1 minute between each 10 pulse. The smear was around 4kb to 650bp. I suspended the pellet in Upstate recipe SDS buffer to 600ul. I should sonicate more-was thinking of trying 20 pulse 3,5,8 times to see what the results look like. Is 20 pulse (~20 sec) at 1.5 power, 40% going to denature protein?
-nellholly-
I sonicate at maximum power for 8 minutes: I do 30 sec on and 30sec off and my ChIPs are ok. I'm using a Bioruptor sonicator. I don't know which one you are using.
-dnafactory-
QUOTE (nellholly @ Jul 17 2006, 11:17 AM)
Hi,
I'm having trouble sonicating-tried 1.5 power, 40% output, 10 pulses 8 times with 1 minute between each 10 pulse. The smear was around 4kb to 650bp. I suspended the pellet in Upstate recipe SDS buffer to 600ul. I should sonicate more-was thinking of trying 20 pulse 3,5,8 times to see what the results look like. Is 20 pulse (~20 sec) at 1.5 power, 40% going to denature protein?
I'm having trouble sonicating-tried 1.5 power, 40% output, 10 pulses 8 times with 1 minute between each 10 pulse. The smear was around 4kb to 650bp. I suspended the pellet in Upstate recipe SDS buffer to 600ul. I should sonicate more-was thinking of trying 20 pulse 3,5,8 times to see what the results look like. Is 20 pulse (~20 sec) at 1.5 power, 40% going to denature protein?
Your proteins could denature if your sonicated material heats up too much. To avoid this, keep your tube on ice or ice/water bath while sonicating.
Regarding your sonication efficiency, if increasing the number of pulses only minimally improves your fragment sizes then try increasing the power (just be carefull to avoid foaming).
-KPDE-