I would like to compare precipitated target DNA in Chromatin Immuno Precipitation by real-time quantitative PCR. How should I compare Ct values of untreated, treated (no antibody) and treated (with antibody) sets taking care of Inputs. How should I plot? Could you please explain? I would appreciate the suggestion of any paper as reference too.
I tried for reamplification (35 cycles) and still there was no product. However I would go for hot-start Taq pol. Could you please suggest the product name and supplier of DNA purification system of your choice, preferentially spin column based. I would like to try that as well.
I am trying to study the methylation status by bisulfite sequencing in genomic DNA from cell lines. I started with 0.5 ug of genomic DNA and after bisulfite modification followed by purification (QIAGEN PCR purification kit), desulfonation, neutralization (with NH4CH3COOH) and precipitaion I dissolved the precipitate in 10 ul. Out of 10, I use 4 ul for PCR amplification.
I used gradient PCR to anneal at different temp (ranging from 48 to 55 C, Tm of the primers are 60 C). After 35 cycle amplification with ThermalAce DNA pol (Invitrogen), I had no detectable product. The primers were designed by using MethPrimer (www.urogene.org/methprimer/index1.html).