QPCR Copy number - (May/11/2006 )
I would like to know what is lowest copy number, determined by real time PCR that can be detected on a gel ? And does it differ from one gene to another?
Thank you very much.
There is no definate answer for this question. Lowest detection limit for a PCR assay is a question varies acroding to your PCR efficienc rather then the target gene. Generally, real-time PCR is more sensitive then conventional one by at least 100 times (base on my experience), due to the nature of the probe.
Meaning that, if you have a know amount of starting template, you can perform a 10x serial dilution, subject each serial diluted template to PCR and real-time PCR (let say SYBR Green)....
you may see you lowest detection limit for PCR is eg. 100,000 copy (on gel), your real-time PCR lowest detection limit should be ~1000 copy (100x lesser).
Efficiency of your assay can be increase through optimization.
Thank you very much for your explanation. I think it fits with my data since I managed to detect 10000 copy number by normal pcr on a gel while I couldn’t detect 100-1000 copy number on the gel.