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in silico primersecondary structure issues.

qPCR; Primer dimer

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#1 tsalihah



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Posted 02 April 2014 - 04:39 PM



 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


dimers (self/hetero):

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?

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