How much template DNA do you use for qPCR - (May/22/2010 )
Hello,
During my initial chip experiments I was not measuring the amount of DNA in each qPCR reaction. I started with same amounts of chromatin during IP and after crosslinking I recovered the DNA in equal volumes of buffer. Out of curiosity I quantified the DNA conc and I have vastly different amounts between samples (not too surprising, really). How does this impact my results? What concentration should I be using?
Thanks for the help -- so far I have some nice prelim results but I am trying to make sure I'm doing everything correctly 
-Chipper-
Chipper on May 22 2010, 09:12 AM said:
Hello,
During my initial chip experiments I was not measuring the amount of DNA in each qPCR reaction. I started with same amounts of chromatin during IP and after crosslinking I recovered the DNA in equal volumes of buffer. Out of curiosity I quantified the DNA conc and I have vastly different amounts between samples (not too surprising, really). How does this impact my results? What concentration should I be using?
Thanks for the help -- so far I have some nice prelim results but I am trying to make sure I'm doing everything correctly
During my initial chip experiments I was not measuring the amount of DNA in each qPCR reaction. I started with same amounts of chromatin during IP and after crosslinking I recovered the DNA in equal volumes of buffer. Out of curiosity I quantified the DNA conc and I have vastly different amounts between samples (not too surprising, really). How does this impact my results? What concentration should I be using?
Thanks for the help -- so far I have some nice prelim results but I am trying to make sure I'm doing everything correctly
You don't need to worry about the DNA concentration in your samples. Just make sure your Cts are within the linear range of your primers and the differences vs. your controls looks good.
-KPDE-