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

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