Toe DNA Preparation For Southern Blots

Toe DNA Preparation For Southern Blots

The DNEasy Kits From Qiagen work well for preparing DNA from toes and tails. Alternatively, the following protocol may be used.
  1. Cut the last phalange of one toe into 0.5 ml of lysis buffer (100 mM Tris pH 8.0, 200 mM NaCl, 5 mM EDTA, 0.2% SDS, 200-500 g/ml Proteinase K) and incubate @ 50-55°C for 24-48 hours with gentle inversion of the tube.
  2. Extract with 0.8 ml of phenol, microfuge 3 minutes. Transfer upper phase to a tube that contains 0.5 ml isopropanol. Cap the tube and invert 7 or 8 times. The DNA will form a wispy clot, that will either attach to an air bubble and float to the top, or will sink to the bottom.
  3. Immediately fish out the clot on the outside of a yellow tip by swirling the tip in the clot. Expel any isopropanol that is in the tip, wait about 3 seconds to allow the remaining alcohol to evaporate, then resuspend the DNA in 100 l of TE in a fresh tube by pipetting up and down about 10 times.
  4. Each toe produces enough DNA for 2 lanes on a Southern. Cut 50 l of DNA in a 200 l volume overnight. EtOH ppt., resuspend and load on gel.

NB: Be aware that Alcide, Clidox and other disinfectants that we are required to use with microisolated mice inhibit Proteinase K activity.