GENtle/Edit primer dialog

GENtle splash screen.png GENtle: Tools and Dialogs

Ligation - Options - Databases - Import - Enter sequence - Sequence editor - Restriction Assistant - PCR troubleshoot dialog - Proteolysis assistant - Projects - Edit primer dialog - Find dialog - Printing - Enzyme management - Sequencing Primers - Silent Mutagenesis - Automatic annotation

This dialog assists in optimizing a primer. For that reason, many variants of the primer are generated and can be examined.

Edit primer dialog.

The center line of the dialog shows the current variant of the primer; details of that variant are shown in the upper right box. OK will end the dialog, committing that variant to the PCR module. Cancel will end the dialog and not change the PCR module. Reset will return the primer in the dialog to the variant the dialog was originally started with.

The list in the lower half of the dialog contains an automatically generated list of variants of the current primer, sorted by an arbitrary score. The "region" of variants can be influenced by multiple settings in the upper left quarter of the dialog. Available settings include:

  • The variation of the 5'-end of the primer to the right and to the left.
  • The variation of the 3'-end of the primer to the right and to the left.
  • The minimum and maximum length of the primer.
  • The minimum and maximum melting temperature of the primer.

Any change of these settings will trigger a recalculation of possible variations. These variations are then evaluated and shown in the list in the lower half of the dialog. Double-clicking one of the variations will change the current variation in the center line, and the properties display in the upper right quarter of the dialog.

Properties displayEdit

This will display:

  1. The primer sequence in 5'→3' orientation
  2. The ΔH and ΔS values
  3. The length and GC contents of the primer
  4. The melting temperature, calculated according to the Nearest Neighbour method (usually best results, but only for longer primers)
  5. The melting temperature, calculated according to the salt-adjusted method (medicore results)
  6. The melting temperature, calculated according to the GC method (simplicistic)
  7. The highest self-annealing score (arbitrary) and the display of that annealing
Caveat : Calculating primer melting temperatures is tricky. If one of the three methods gives a totally different result than the other two, ignore it. Also, the melting temperature is only calculated for the 3'-end of the primer that anneals with the sequence!