Hello,

I've been having issues with my standard curve and wonder what the possible issue with it could be.

• plasmid standard with starting concentration determined to be 2E8 copies/ul
• previous runs confirm this copy number
• I serially diluted the plasmid 5 fold from 2E6 to 640 copies/ul
• When run on qPCR, standard is skewed way off with fold changes ranging from 11 to 200
• Primers and probes work fine with other standards and controls
• user error was checked and dilutions have been double checks in respect to calculations

Could it be due to plasmid degradation leading to nonspecific binding that's causing such skewed numbers? Can anyone speak to what may be the root cause of this?

Thank you!