Whole mount in situ hybridization to study gene expression during mouse development.

Set up a restriction reaction to linearize the plasmid DNA. Check for complete digestion, then incubate with 200 μg/ml PK in 0.5% SDS at 37 o C for 60 min. Extract with phenol/chloroform, and recover by ethanol precipitation. Wash the pelleted DNA with 70% ethanol; redissolve in Rnase-free TE buffer at a concentration 1mg/ml. Set up a labeling reaction according to the RNA polymerase instructions in total volume 20 μl. Incubate @ at 37 o C for 60 min. Add 2 μl of 0.5M EDTA (pH=8.0) and 4 μl of 4 M LiCl, mix, then add 120 μl of 100 ethanol and put at 20 o C for a few hours. Pellet the labeled RNA at max speed in a microfuge at 4 o C for 30 min. Wash the pellet with 70% ethanol, dissolve in 20 μl of Rnase-free water. Examine 1 μl on 1% agarose gel.