I am trying to optimize a TaqMan/Gene Expression like assay that targets three different genome areas in one assay. Because I am technically adding only three reagents (1 reagent = primer for and rev and probe plus the obvious Universal Master Mix and genomic DNA) thus the primer and probe concentrations are fixed in the sense that the reagent has the concentrations already set and I can only adjust for amount of reagent to add. I have been able to get the assay to work however the efficiency aka the slope is not optimal for the standard curve (-4.62 for target 1, -4.60 for target 2 and -3.83 for target 3). I was wondering if anyone has any suggestions in getting the slope to the optimal range of -2.8 to -3.6. The Master Mix I am currently using is the following:
Universal Master Mix 12.5 ul
Target 1 @ 10X .5ul (this includes primer and probe in one reagent)
Target 2 @ 10X .75ul (this includes primer and probe in one reagent)
Target 3 @ 10X 1ul (this includes primer and probe in one reagent)
DNA 4 ul
I am thinking of increasing the amount of Unviersal Master Mix to increase the amount of Taq present in the reaction. But any other suggestions would be greatly appreciated.
Thank you in advance!