temperature estimated absorbance coefficients inter molecular self complementarily estimation and intra molecular hairpin loop formation http www basic northwestern edu biotools oligocalc html http www idtdna com analyzer applications oligoanalyzer default aspx the sequence of the bases are not shown complementary in order to prevent taq polymerase from extending the substrate dna until the ape cuts the substrate dna said another way the strands are complementary except for the 3 u2032 end of the substrate and was designed with a 3ddc 3 u2032 block and is non complementary to prevent taq polymerase from extending the substrate molecule until it is cut by the ape to perform the ape activity assay 1 ul of 100 um substrate oligonucleotide was hybridized to 1 ul of 100 pm template oligonucleotide by briefly vortexing at room temperature to this 1 u of ape1 1 ul of 10 u00d7 reaction buffer and 6 ul of molecular grade water were added the reaction mixture was incubated for 1 hour at 37 u00b0 c then a 5 ul aliquot was added to the pcr reaction mixture consisting of 12 5 ul bio rad master mix 1 ul forward primer 1 ul reverse primer 1 ul probe and 4 5 ul water after determining that the substrate and the template oligonucleotides efficiently hybridize to one another the reagents where cycled separately and in combination without addition of ape to ensure that false amplification would not occur the final two reaction conditions contained a substrate oligonucleotide referred to.