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Bead saturation


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#1 samit

samit

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Posted 04 October 2010 - 04:07 AM

Hi all,

I am new user in bioforum as well as I have recently joined the lab as a PhD student. i have been doing ChIP experiment.I am using Sepherose A beads for the pull down of nonspecipic protiens, My colleague told me to saturate the beads by washing it with sonication buffer. And to use this bead at the time of preclear.
I added the sperm DNA and the BSA to the samples to be precleared and then added the washed beads to the sample and kept it for 1 hour at 4 degree for incubation. I used 70 ug of DNA to prepare my input samples, IPs and the one without antibody. I made qPCR analysis after washing and DNA isolation, I found that the values of samples without antibody are coming greater than the values of my IPs.

Can anyone please help me to come out of this problem,

Thanks in advance,




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