Kras-LA1 & LA2 Southern Strategy

This protocol is used to distinguish between KRas LA1 and KRas LA2 as the PCR protocol cannot.

5'internal xba/sal probe

Digest with Hind 111(#798 983) and Asp 718(#1175 050) both from Boehringer Mannheim.

Wash conditions:

Wash 3 times at 68C (max) each for 20-30mins in
2ml  0.5M EDTA
20ml 1MNaPo4
50ml 20% SDS
928ml  water

WT   10kb
5' Mut 8.9kb
3'Mut 6.1kb
Activation of Kras in lungs should be shown by loss of intensity of the 5' mutant bands.

Controls:

WT DNA (10kb only)
Lat 44 DNA (10kb, 8.9kb, and 6.1kb bands)
Act DNA (10kb and 8.9kb bands, the recombination was allowed to occur in vitro)