Posted 26 April 2012 - 09:56 AM
It's now two months that I've been stuck in the shearing part of the ChIP assay. I'm using cup horn settings but I always have lots unsheared DNA and a perfect smear around 400bp. I have gone from 1 min of sonication to over 40 min but I still do have that unsheared part. I have tried anything that the company suggested: polystyrene tubes, decreasing the sample volume, distance from the horn, etc. However, it is still a big mess. I'm really exhausted and don't know what to do. I would be very very very thankful if any of you have any suggestion.
Ps. I'm doing ChIP on MCF-7 cells
Posted 27 April 2012 - 07:28 AM
Posted 30 April 2012 - 12:03 PM
Posted 30 April 2012 - 12:07 PM
Posted 30 April 2012 - 12:35 PM
I use 10 ul of my sonicated sample and add 40 ul IP elution buffer (50 mM NaHCO3 & SDS 1%).
Then I add 2 ul of 5 M NaCl
I boil them for 15 min
then I add 5 ul of 3M NaOAc ph=5
and then I purify them with PCR kit
Posted 30 April 2012 - 12:43 PM
Posted 30 April 2012 - 12:47 PM
Posted 30 April 2012 - 12:53 PM
Posted 01 May 2012 - 07:52 PM
should not you use polyPropylene tubes(Non_Sticky)?
Edited by memari, 01 May 2012 - 07:52 PM.
Posted 02 May 2012 - 11:28 AM