I am very new at designing primers and qPCR and I need all the help I can get.
I am designing primers using idtdna primerquest (for qPCR and SYBR Green) and run the subsequent primer pairs on the oligoanalyzer for secondary structure.
I use the following rules to rule out primer pair (for presence of secondary structure in silico):
1.3’end pairing : ∆G≥ -2 kcal/mol
Internal pairing : ∆G≥ -3kcal/mol
1.3’end pairing : ∆G≥ -5 kcal/mol
Internal pairing : ∆G≥ -6 kcal/mol
and for hairpin and dimers, Tm of secondary structure atleast 5oC≤ Ta primer
but the end result left me with no primers at all. I know that anything ∆G≥-9 kcal/mol is also acceptable, could I make exception for dimers that had -6 to -9 kca/mol? or should I just cut my loses design a whole new batch of primers?