High Ct Values - (Apr/23/2007 )
I've read many topics and realized what a bright bunch you all are, so I decided to seek your help
I've started doing real-time PCR of genomic bacterial DNA using TaqMan probes and running the samples on an Bio-Rad iCycler. So far, my standard curve comes out pretty well, with correlation ~0.99 or greater, and the efficiency is usually around 100-120%. The problem is that the Ct values start around 26 for the most concentrated sample (calculated copy number 10^6) and I have to run 50 cycles to get the more dilute standards. I cannot consistently detect below 100 cells/sample.
I've played around with MgCl2, probe and primer concentrations and they are all currently a little high at 5 mM (MgCl2), 1.125 uM (primers) and 0.3 uM (probe). The DNA template is relatively pure, going by spec analysis, so the starting concentration is not off I think.
I realize there are probably many factors I'm overlooking, but any suggestions?
why can't you detect below 100 genomic copies? Do your NTCs amplify unspecific by-products like primer dimers? And did you make your standard curve with genomic DNA or did you use PCR products or plasmids? Were these standards able to be diluted reproducible below 100 copies? I do ask, since I once experienced problems with a standard, containing SNPs in the primer sequences. The amplification efficiency from the standards was much higher than the test samples. And perhaps, your standards are quantified wrongly, since none of your samples contains less DNA as 100 genomic copies.