To prepare for a set of serial dilutions of qPCR standards I follow an online protocol that assumes using either gDNA or a plasmid. You then identify the mass of DNA per genome and divide by copy number and away you go with the subsequent calculations. But my problem is this....if I were to use synthetic standards ie; custom oligos that are surrogates for the PCR product......they are short in length and therefore do not relate at all to the genome of interest in terms of "mass of DNA" .....is this a problem and if so is there an alternative protocol for the calculations? I am using the Applied Biosystems online protocol presently.
I guess my question even extends to using a plasmid with the PCR product inserted as it bears no resemblance in mass to the genome from whence it came. It has bugged me for sometime.