Toe DNA Preparation For Southern Blots
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The DNEasy Kits From Qiagen work well for preparing DNA from toes and tails. Alternatively, the following protocol may be used.
- 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 µg/ml Proteinase K) and incubate @ 50-55°C for 24-48 hours with gentle inversion of the tube.
- 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.
- 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 µl of TE in a fresh tube by pipetting up and down about 10 times.
- Each toe produces enough DNA for 2 lanes on a Southern. Cut 50 µl of DNA in a 200 µl 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.
