Molecular Beacon Primer Design Protocol

1. Enter exon sequence.

2. Restrict primer temperature to 59-61ºC.

3. Restrict primer length to 19-21 base pairs.

4. Change the following parameters:

a. Hairpin Max Delta G (3’end) - 2 kcal/mol b. Hairpin Max Delta G (Internal) - 3 kcal/mol c. 3’ end max delta G - 10 kcal/mol d. Self Dimer Max Delta G (3’end) - 5 kcal/mol e. Self Dimer Max Delta G (Internal) - 6 kcal/mol f. Run/Repeat (dinucl.) Max Length - 3 bp g. G/C clamp-Target consec. G/C’s at 3’ end – 1 h. Amplicon Length – 65-75 base pairs i. Max Ambiguous Bases in Amplicon – 0 j. Max Primer Pair Tm Mismatch – 1C k. Cross Dimer Max Delta G (3’ end) – 5 kcal/mol l. Cross Dimer Max Delta G (Internal) – 6 kcal/mol m. %GC Content – N/A