2 replies to this topic

[taurean12]

I apologise in advance if this is the wrong place to post a question. I was looking at many different protocols for ChIP on line. Does it make a big difference if one prepares the protein A, salmon sperm DNA in advance

or can one add the washed beads and salmon sperm DNA directly to the sonicated lysates for an hour for pre-clearing.

Thanks

[pcrman]

I don't quite understand you, but It is OK to prepare protein A, salmon sperm DNA in advance in advance and store it at 4C for future use.

Yes, after sonication and have dilute the lysate with ChiP dilution buffer, before adding antibody, you have to preclean your sample with protein A, salmon sperm DNA in advance otherwise you will get false positive signals.

[taurean12]

Thanks for your reply. Could you give me a protocol for preparing salmon sperm DNA protein A beads and how much should I add for preclearing?

Sorry if my question was not clear enough. One protocol said

' Perform immunoclearing by incubating 1ml of soluble chromatin with 2 ug of sheared salmon sperm DNA , 20 ul of pre-immune serum and 45 ul of 50% protein A sepharose for 2 hrs at 4oC.'

The other one suggested making the salmon sperm /protein A beads ahead of time for 45 mins before adding to the chromatin lysates. I was wondering if both of these protcols are acceptable

Thanks