Collecting Ear punches:

1.  Mice can be anesthetized or non-anesthetized for punching.  If you have to do a lot, then anesthesia may help the processes move more quickly.

2.  Ear punch material can be collected into eppendorf tubes for DNA extraction using forceps.  Clean instruments and area with dilute bleach between each punch to prevent cross-contamination.

3.  Store ear punches at -20oC or process immediately.  DNA degradation may occur if the specimens are left at RT.

Processing ear punches.

Digestion mix: For 100ul rxn

50mM Tris pH 8.0 5ml 1M Tris pH8.0

2mM NaCl 40ul 5M NaCl

10mM EDTA 2ml 0.5M EDTA pH8.0

1% SDS 10mls 10% SDS

+82.96 ml MQ HOH

Add to final concentration of 1mg/ml Proteinase K at time of digestion. 

1.  Add 20ul of Digestion mix plus 1ul of Proteinase K stock (20mg/ml, fridge next to Sana’s bench).  Make sure the punch is at the bottom submerged in solution.

2.  Place tubes in 50oC heating block for 20min.  Vortex.  Incubate at 50oC for 20 more min.

3.  Remove tubes from heating block.  Add 480ul of water.  Vortex.  Place in 100oC heating block for 9 minutes to inactivate Proteinase K. 

4.  Store at 4oC to -20oC depending on speed of use.  Mix well before taking DNA for PCR rxn.