Ampification in no-antibody control of ChIP assay
Posted 27 December 2004 - 12:45 AM
Merry Chrismtas and happy New year.
I am working on Chromatin immunoprecipitation now. I am so upset that my no antibody control also has PCR band. Did anybody meet this kind of problem? What should I do? By the way, I use the ChIP kit from Upstate. I did no template control when I did PCR. There is no band. So it is not because of PCR contamination. Thanks
Posted 27 December 2004 - 12:05 PM
Posted 29 December 2004 - 11:53 AM
Posted 29 December 2004 - 03:43 PM
As I had mentioned, you may be experiencing non-specific binding of proteins to the salmon sperm/protein A agarose. To pre-block the agarose, please perform the following:
Incubate/wash the Salmon Sperm DNA Agarose before using it. It can be blocked in 1-5% BSA and Chip dilution buffer to "block" the agarose matrix (you can incubate for 30 minutes mixing at room temperature). After incubation, spin the agarose and remove the 1% BSA/ChIP assay buffer supernantent. Wash once in ChIP assay buffer and continue with the assay as normal.
BTW, I usually get no amplifications in no-antibody controls even without prewahsing.
Hope that helps.
Posted 04 January 2005 - 08:53 PM
Edited by seenew, 04 January 2005 - 08:58 PM.