Biology-Online • View topic - Improving qPCR assay detection limits

Join for Free!
121820 members

Improving qPCR assay detection limits

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderator: BioTeam

Improving qPCR assay detection limits

Postby Hriss » Thu May 12, 2011 10:06 am

Hi guys,
I am trying to design a real-time PCR assay for copy number determination of an antibody gene in mammalian cells, using TaqMan fluorescent probes.
The issue is, I need to find a way to improve the sensitivity of the assay. So far, it works perfectly in the range 10^8 – 10^4 copies of either antibody chain per reaction (acceptable efficiency, Pearson correlation, amplification is every three cycles). When 10-10^3 standards are added to the standard curve, however, they all cross the threshold line at about 24 cycles along with the 10^4 standard, irrespectively of the fact there is far less DNA in them, and they should cross the line a lot later.
So, far, we have tried optimising primer concentrations and cycling conditions, tried different primers, ran the assay on a different machine…. The problem still exists.
Any bright ideas or suggestions on what to try next?

Posts: 1
Joined: Thu May 12, 2011 10:03 am

Return to Molecular Biology

Who is online

Users browsing this forum: No registered users and 0 guests