( but the program will not proceed witho...)Real-Time PCR: General Considerations, Rev A

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6. Click on the button marked Fold DNA next to the smiley face
7. Now youll get a list of structures ideally you will see only one. Pick a format such as PNG to view the structure.
8. The T_m of the structure, which appears in a separate window (Loop Free-Energy Decomposition), will tell you at what temperature this structure will form.

Primer Design
The goal is to design primers with a T_m higher than the T_m of any of the predicted template secondary structures. This ensures that the majority of possible secondary structures have been unfolded before the primer-annealing step. Follow these parameters when designing primers:
Design primers with a GC content of 5060%
Maintain a melting temperature (T_m) between 50 and 65C
Eliminate secondary structure
Avoid repeats of Gs or Cs longer than 3 bases
Place Gs and Cs on ends of primers
Check sequence of forward and reverse primers to ensure no 3' complementarity (avoids primer-dimer formation)
AdjusT_ment of primer locations outside of the target sequence secondary structure may be required
Verify specificity using sites such as the Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov/blast/)

Reaction Components and Conditions
Components
Optimization conditions can vary with assay type. Therefore, these conditions should be considered when establishing a new assay:
MgCl₂ concentration (3.06.0 mM)
dNTP concentration (200600 M each dNTP)
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