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How can I determine if a hairpin in my oligo is too strong to allow hybridization?
Using free OligoAnalyzer® software, part of the IDT SciTools programs, enter your oligonucleotide sequence and choose “Hairpin.” The software will generate a series of possible hairpin structures. You can arrange these structures in order of decreasing melting temperature (Tm). If the highest hairpin Tm is at or above your annealing temperature, that hairpin is likely to impede hybridization.