Sean Lauber:Kras G12D Genotyping

This protocol makes use of the DirectPCR mouse tail lysis reagent (Viagen, cat 102-T) to obtain template and the

hot start Platinum Taq (Invitrogen, cat 10966-026). 1 mm tail ends are collected from mice (typically at 4-6 weeks)

and the tail is degraded using the DirectPCR reagent before use in PCR.

1. 125 ul of DirectPCR reagent and 4.73 ul of Proteinase K are added to each tube containing a 1 mm tail snip 2. The tubes are heated at 55*C O/N 3. The tubes are heated at 85*C for 45 min to inactivate the proteinase K 4. After vortexing briefly, 0.5 ul of this template is added to a PCR reaction along with the other reagents:

 FOR EACH PCR REACTION:
 2.5 ul   10X PCR buffer
 0.5 ul   10 uM dNTPs
 0.75 ul  50 mM MgCl2
 1 ul     primer 74B*
 1 ul     primer 73B*
 0.5 ul   template (from DirectPCR)
 0.1 ul   Platinum Taq
 17.75 ul sterile water
 --------
 25 ul  
 *primer 73/74B are mixed together, so add 2 ul of this mix

5. PCR is then performed with the following parameters:

 94*C  3 min

 94*C  0.5 min