This protocol was developed by Jon Neumann at the University of Cincinnati

• Ear clip the mouse. Take the tissue and put into a microcentrifuge tube. Clean off clippers carefully between animals.
• Digest each clip in 100 � of 1XPCR buffer with added detergents (0.45% NP40, 0.45% TWEEN 20) and 10� Proteinase K (10 mg/ml) @ 60o C for 2 hrs. to overnight. If using a short incubation time, vortex several times during the incubation.
• Denature the Proteinase K by boiling for 15 min. (DO NOT BOIL FOR ANY LESS THAN 10 min.; Reboil prior to any subsequent analysis). Cool on ice for 5 minutes.
• Aliquot 18 � of PCR reaction buffer into a PCR tube
• PCR Reaction Buffer
  • 1X PCR buffer
  • 2.5 mM MgCl2
  • 200� dNTPs
  • 1� each primer
  • 1 Unit Taq Polymerase
• Add 2 � of ear digest to the tube; mix by pipeting.
• Overlay with 30-40 � light mineral oil (if not using hot top PCR machine)
• Put into cycler and run the following program:
  • Hold @ 94o C for 4.5 min.
  • 30 X Step cycles of: 94o C for 30 sec.
  • required annealing temp. for 20 sec. (varies according to G/C content of primers)
  • 72o C for 1 min.
  • Hold @ 4o C until ready to analyze.
• Add 2 � of agarose dye mix to each tube, and load all onto a 1.5-2% agarose gel.