Forward: CAAGGGTATCACCTTCAAAC

Reverse: CTTCAAACCAAGGGTATCAC

Bold - primer, underlined - backbone

Parameters of priming sequences:

Criteria Valid values Forward Reverse
Gibbs free energy (kcal/mole) -40 - -30 -35 -33
Melting temperature (°C) 50 - 65 52.4 51.11
Pimer length (nt) 18 - 25 21 22
GC-content (%) 20 - 80 50 40

Parameters to exclude in whole primers (if all/if any):

Criteria Treshold values
Min Gibbs free energy (kcal/mole) -7
Max melting temperature (°C) 20
Max complementary motifs length (nt) 3

Forward primer self-dimers:

Delta G: -6.6 kcal/mole Base Pairs: 4

CAAGGGTATCACCTTCAAACTT
       ||||  ::  :::: 
TTCAAACTTCCACTATGGGAAC

Reverse primer self-dimers:

Delta G: -6.6 kcal/mole Base Pairs: 4

      CAAGGGTATCACCTTCAAACTT
       ||||  ::  :::: 
TTCAAACTTCCACTATGGGAAC

Hetero-dimers between found primers:

Delta G: -9.5 kcal/mole Base Pairs: 6

GATGGTGATGTTAATGGGCAC
      :: ||||||     ::
TTCAAACTTCCACTATGGGAAC

Hetero-dimers between the forward primer and other sequences in PCR reaction:

Delta G: -9.5 kcal/mole Base Pairs: 6

GATGGTGATGTTAATGGGCAC
      :: ||||||     ::
TTCAAACTTCCACTATGGGAAC

Hetero-dimers between the reverse primer and other sequences in PCR reaction:

Delta G: -9.5 kcal/mole Base Pairs: 6

      GATGGTGATGTTAATGGGCAC
      :: ||||||     ::
TTCAAACTTCCACTATGGGAAC