Reaction Mix

  • 10X Perkin Elmer Buffer II 1X
  • 25mM MgCl2 2mM
  • 2.5mM dNTPs 0.2mM
  • 20 uM oIMR013 0.75 uM
  • 20 uM oIMR014 0.75 uM
  • 20 uM oIMR015 0.5 uM
  • 20 uM oIMR016 0.5 uM
  • DNA loading dye* 0.138ul per ul total volume
  • 5 Units/ul AmpliTaq DNA polymerase 0.014 Units/ul
  • DNA 10ng-200ng/reaction

Temperature Cycling

  • 94°C 3minutes
  • 94°C 20 seconds
  • 64°C 30 seconds
  • -0.5°C/cycle
  • 72°C 35 seconds
  • Goto Step #2, 12 times
  • 94°C 20 seconds
  • 58°C 30 seconds
  • 72°C 35 seconds
  • Goto Step #7, 25 times
  • 72°C 2 minutes
  • 4°C forever
Note: This assay generally works with any primer with a Tm near 60°C (57°C-63°C). However, because of nonspecific background bands generated by neo primers, always run a known neo negative and neo positive sample for comparison