Note: This topic is not applicable to BAM-based projects.
Primer Walking parameters control the characteristics of primers for contig extension and coverage.
Primer Walking parameters are specified by going to Project > Parameters and choosing Primer Walking from the list on the left.
•Minimum Length - The shortest acceptable primer length in bases.
•Maximum Length - The longest acceptable primer length in bases.
•Salt Concentration - The millimolar concentration of salt (Na) at the start of polymerization. Increasing salt concentration raises the primer melting temperature.
•Formamide Concentration - The concentration of formamide expressed as a percentage in solution. Increasing formamide concentration lowers the primer GC melting temperature, as computed by the GC content method. The formamide setting has no effect on the nearest neighbor calculation of melting temperatures, which is the method used for primer selection.
•Temperature for Delta G calculations - The entropy calculations for the base temperature used in the delta G calculations for thermodynamic modeling. Changing the base temperature will affect the delta-G calculation presented in the report.
•Avoid Hairpins – When selected, SeqMan Pro will avoid selecting primers that fold and bond to themselves.
•Avoid Dimerization – When selected, SeqMan Pro will avoid selecting primers that hybridize to themselves.
•Ignore Duplexing this distance from 3’ - Enter the distance (in bases) from the 3' end of the primer where hairpin formation and self-hybridization may be ignored.