How well does ccPCR indicate the presence of background binders?

I started carrying both + and - protein selected RNA to ccPCR at R3. The ccPCR gel images can be seen here. R3 showed nearly identical amplification between + and - protein. R4 seemed to show a reduction in background binders through the later amplification of - protein selected DNA. Finally, R5 showed nearly identical amplification of + and - protein again. I was wondering how clear of an indicator this is as to the current composition of the selected pool. If we are still performing binding assays after R6, I am not to far from determining the real binding of my pool, but I was wondering if the evidence from the ccPCR gels is enough to go ahead and split of another selection from R4, the round that seemed to show a reduction in background binding.


Nia_Fernandez said...

by " - " do you mean a negative control? a selection done without any protein present? If this is the case then I think its related to DNA binding to the filter. This should decrease as you do more rounds with negative selections. I hope this helps

Michael Ledbetter said...

Yeah the "-" means the RNA collected from the filter without the presence of protein. I know that it should decrease through the number of rounds, but I was concerned by the fact that the background binding seemed to decrease in R4 but then reemerge in R5. I spoke with Brad about it, and he said that, when I perform a binding assay after R6, I should assay R1, R3, R4, and R6.