i try to establish a sybr q pcr wich detects two different genes. i´ve already cloned my genes and a standard dilution was done. Followed by optimization of MgCl2 and pimer concentration.
Due to the reason that i try to quantify aqueous bacteria via this two genes i designed and IC which runs with the same pcr conditions.
But my question is:
I calculate via the cp of my standard dilution my concentration of desired genes. Ok, but if there is an influence like inhibition due to the seawater i can detect this via my IC. So how would you calculate the inhibition of the desired genes in the sample in relation to the IC.
Standard 1E2 concentration with Cp 24; sample 1 5E1 concentration with Cp 27.
IC in a.d. Cp 24; IC in environmetal sample Cp 30.
This means a delay of 6 Cycles of the IC.
So there is an influence visible but how should i implement this delay in final sample concentration calculation?
Thanks for your help