Background in TF-ChIP PCR - Is it ethical to optimize it away? (Aug/10/2005 )
In papers, it looks like everyone gets a completely clean background reaction. This has concerned me in the past, but based on some of the posts here I am no longer convinced that it is wrong to get a band in the negative control.
Maybe the authors(of the above-mentioned papers) could have used tricks of PCR to eliminate any visible amplimer in the negative control, I don't know if I agree with this tactic, it seems a little like manipulating data, but if your background is 0.1% and your enriched fraction is 1% then using conditions where your limit of detection by PCR is 0.2% you can do an experiment that looks like there is no background but is really due to using less sensitive assay conditions.... This can also be accomplished by manipulating the exposure time of the gel...
Anyway, does anyone have a comment on this? Is it right to optimize your assay to eliminate a visible background band?? 
i think the target gene/region PCR assays should first be optimized as if one were simply trying to develop a good PCR assay (this is often done with something like the input DNA).
however, if the PCR assay is suboptimal in some way, whether it be composition of the reaction, or thermal cycling conditions, one would expect all the templates' reactions (specific IP and mock/background control) to be affected. so in a ChIP that worked well, an optimized PCR test may give amplicons in the specific IP at say 30 cycles, and in the mock/background at 40 cycles. if the same ChIP templates are instead run under suboptimal PCR conditions, the cycles may shift to 35 and 45 respectively.
if one cycles long enough, i think you'll eventually always get a band in the mock/background control.
so in conclusion, i believe one ought to optimize each PCR assay to begin with, but if they do happen to be suboptimal, it may not be so bad.
Maybe the authors(of the above-mentioned papers) could have used tricks of PCR to eliminate any visible amplimer in the negative control, I don't know if I agree with this tactic, it seems a little like manipulating data, but if your background is 0.1% and your enriched fraction is 1% then using conditions where your limit of detection by PCR is 0.2% you can do an experiment that looks like there is no background but is really due to using less sensitive assay conditions.... This can also be accomplished by manipulating the exposure time of the gel...
Anyway, does anyone have a comment on this? Is it right to optimize your assay to eliminate a visible background band??
Although not sure how many cycles for your PCR , I dont think, as high as 45 cycles is good number.
Too high cycles , risk out of limitation of PCR ?? It's better to increase your template and reduce the cycles???
Different polymerases have different efficiencies, try some good polymerases and you can reduce cycle number without increasing template.