ChIP: Equal DNA Pull-Down in Positive and Negative Controls - HELP - (Sep/11/2006 )
Haven't tried that yet. Seems like it would reduce both the positive and negative signal, not necessarily just reduce backgound. I may go to the kit you're using if I can't get the background better. I'm also getting very low quantities, i.e., my Ct during qPCR is in the 38 range for the positive sample. From what I'm seeing in this forum that's pretty low for a ChIP-Chip.
also, styounger, I noticed in another forum you mentioned doing serial dilutions in order to reduce Ct. Did this work?
Serial dilutions didn't help. The Ct values got higher for both positive and negative samples, but the difference between the two didn't improve. For my experiments I'm harvesting roughly 20 million cells per treatment. I resuspend the harvested nuclei in 1 ml of lysis buffer. Of that 1 ml, I use 25 ul as input and 200ul per pulldown. That translates to about 4 million nuclei equivalents per pulldown (granted I probably lose a lot during harvest and nuclear isolation). On an average experiment (for GAPDH promoter DNA) my input Cts are around 22, my negative control IgG is around 34, and my RNA Pol II is around 27.
You should use same amount of negative control IgG from same species, should include a positive control. The other way is to do PCR with a negative control gene promoter and to subtract from the negative control gene promoter, then calculate fold enrichment after subtracting from negative control promoter. I used a fast Chromatin IP kit from {edit} and found it to be very simple and fast (4 hours) with much less nonspecific issues. It is in strip well format and can be done in high throughput.
If you are interested in doing the 96 well ChIP without paying for the kit we published almost exactly the same protocol in Nucleic Acids Research in January (the main difference is that we don't transfer anything out of the plate and we speed up the IP by an hour using an ultrasonic bath). We also make our own protein A plates (extremely simple and cheap). Here's the link to the article:
http://nar.oxfordjournals.org/cgi/content/full/36/3/e17
If you're interested I can also give you a detailed protocol.