Dear all,

I hope you could help me with the following question: There are different adjuvants that are used in qPCR for quantifying soil microbial functional genes (which often means using »degenerate« primers with wobble bases), like for example T4gp32 protein, BSA, DMSO etc. I know each of those have it's unique properties and sometimes BSA and DMSO are also used in combination. As far as I've understood BSA is used to reduce inhibition but also to increase specificity at amplifying regions with secondary structures while DMSO binds to the DNA and prevents reannealing of single stranded DNA and also facilitates primer annealing so therefore also increases specificity. T4gp32 should also increase the yield and specificity of PCR products from soil samples. Are my conclusions correct? If yes, my question is the following: How can I design a decision tree on whether to use any of the mentioned adjuvants in qPCR reaction for quantifying soil microbial community using a standard curve? If there is some paper where those things are nicely explained please let me know. Some examples from our lab are listed below (4 different examples where T4 gp32 protein was used):

I know that inter-run variability can be quite high and copy numbers of the same samples can vary up to 30% so maybe I'm stressing too much here, but I would really like to understand the mechanisms involved in the reactions.

To sum up: It seems that T4gp32 influences different assays in different ways. On one hand it looks like it increases specificity but also decreases efficiency of the assay, on the other hand it doesn't influence the efficiency much, but lowers or increases the copy numbers of the samples. How could I decide for its use (or any of the other adjuvants for that matter)? Probably a gel electrophoresis would be a good start to examine the products there (eventhough melt curves of all the assays were specific)?

Just to note: All of the above tests of T4gp32 addition were done in the same run for individual assays. Melt curves were always specific with only one peak. Inhibition was tested by spiking the external plasmid of known quantity to the sample amount (2ng DNA) that is usually used for qPCR reaction and comparing the Cq cycles with positive control consisting of only water + known quantity of external plasmid. No inhibition in any of the samples was noticed.

I know it's rather exhaustive read but I'll really appreciate all the answers that I get.

Thank you very much in advance!