I observe extremely high Cts for the immunoprecipitated sample (positive locus) and for my internal controls comparing with the Cts for the input DNA. What does it mean?
Analyzing your description we can conclude that the PCR did work but the DNA recovery from the immunoprecipitation was too low. Low enrichment is also confirmed by the high Ct values for the controls. To avoid the problem of low IP recovery: • Use the correct concentration of antibody as recommended in the manual. • Double check the amount of DNA and antibody • Use the recommended ratio of antibody to DNA – the ratio is critical. • Always use fresh dilutions of antibody (within the same day). • Avoid excessive freeze-thaw of the antibody – you may aliquot the antibody upon receipt, freeze the aliquots, and prepare fresh dilutions before each use. • Assure that the purification of the immunoprecipitated DNA is as recommended in the manual.
