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Sonication optimization using Cup horn


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#1 flare

flare

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Posted 09 June 2009 - 10:20 PM

Hello!

I am conducting a Chip assay with HepG2 cell, following the Upstate Chip assay kit protocol.
It's my first time to do this experiment, so I guess I might have to go through many trials and errors.
But I wish I could find anyone who can help me minimizing that.

My problem occured at sonication step.

I am using Vibra Cell sonicator VCX-500/750 (Sonics) with a cup horn.
Before sonication, I aliquoted the resuspended cell with 1X10^6 cell / 200ul concentration in eppendorf tube.

Sonication was performed at 22kJ ~ 100kJ energy with 5 X 10 s, 10s interval, or 5X 30s, 30s interval. All at 30% amplitude.
However, the sheared DNA sizes of different samples were almost same; the average size was approximately 1200 bp.

My question is :

1. What is your advice? Should I raise the sonication energy? or time? which would be more conclusive?

2. If you are using a cup horn method, can you provide me your own sonication condition?
(especially with your sonication energy and time)

Thank you.

Edited by flare, 09 June 2009 - 11:24 PM.


#2 epi123

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Posted 21 June 2009 - 07:54 AM

hi there,

i don't use cup horn, however i've found it helps to try sonicating 1X10^6 cell / 50 microliters concentration in eppendorf tube.




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