Nonspecific precipitation in ChIP - (Jan/27/2006 )

Hi everyone,

one problem haunts me these days in my ChIP assay. Everything is OK in my ChIP: the sample with antibody has correct band, the negative sample has no band, BUT, one sample without antibody has band always!! I extended the wash time, all the band disappear, including the correct and the wrong band; I shortened wash time, all band appear again! Ohhh, , i have no idea how to modify it now. Could you give me some advice?

btw, i use the selfmade solution in ChIP.

THANKS A LOT!!

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Do you block the beads with non-specific DNA (salmon sperm DNA) prior to running your IP.

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O, shall I? I did not add any non-specific dna, but only protein A sepharose slurry 30mins, and then antibody overnight, and then protein A+G sepharose slurry. I noticed that these non-specific dna is not contained in many protocols. It is necessary? I will try it. Thank you.

Could you tell me the volume of salmon sperm dna, or give me one copy of your protocol? really appreciated.

wszyy

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Yes, standard protocol doesn't include the preclean step. If there is a problem, precleaning may help. Check this post for protocol
""../../forums/inde...findpost&p=9657

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Thanks a lot! This really helps.

BTW, today is Chinese New Year, Happy new year to you guys.

Wszyy

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