Avoid Shearing of the DNA by limiting mixing to inversion and gentle shaking. Vortexing of samples is not required to perform the following protocols.
Avoid contamination by rigorously cleaning pipetman and using autoclaved tips during manipulation of genomic DNA and reagents to be used with genomic DNA.
1. Mice are toe-clipped and approximately 0.5-1 cm of their tail is snipped off and placed into a 1.5-ml microcentrifuge tube. Digestion is improved if the tail piece is cut into several smaller pieces. This tail can then either be processed immediately or frozen at -20íC. Under no circumstances should tails be left at room temperature. If the mice are younger than 21 days, they can be toe-tagged and the tail cut for analysis.
2. To each tail, add 600ml of tail digestion buffer (which has been supplemented with proteinase K at a final concentration of 0.5 mg/ml) and place at 55íC for 6-24 hours. OK to use epindorf repeater.
Mixing during the digestion period will decrease the time required to digest the tail sample and improve DNA quality.
Tail digestion buffer (w/o proteinase K):
- 315 ml of autoclaved deionized water
- 25 ml of 1 M Tris-HC1 (pH 8)
- 10 ml of 5 M NaCl
- 10 ml of 0.5 M EDTA (pH 8)
- 50 ml 10% SDS (or 10X SET)
- Prior to use, 50 ml of proteinase K (10 mg/ml) is added for each ml of digest buffer
3. Once the tail material is digested, each sample is extracted once with 600 Ál phenol:chloroform (75:25) (add with repeater, invert tubes multiple times to mix or vortex). Centrifuge the samples at 13,000 RPM (max) for at least 5 min. to separate the organic and aqueous phases, the aqueous (top) phase is transferred to a new macrocentrifuge tube (use a wide bore pipette tip). Do not try to transfer the entire aqueous phase to a new tube. Do not transfer any phenol/chloroform.
4. Extract the aqueous sample with chloroform (600 Ál, mix well as noted above), but this time instead of transferring the aqueous phase to a new tube, remove the organic layer from under the DNA sample. It is okay to leave small droplets of CHCl3.
5. Add 1 ml of 100% ethanol (-20íC) and mix the tubes by inverting them several times (Make sure tubes are very well mixed). The DNA should form a visible mass. Pellet the DNA by spinning at maximum speed for two minutes. Carefully pour off the ethanol and wash with 70% ethanol/0.1 M NaCl (-20íC). Mix briefly, then centrifuge at max RPM for two minutes. Carefully pour off the wash and inverted to dry for several minutes. (Watch each pellet and do not let any DNA slide out of the tube!!!). Depending upon the pellet size, the DNA should then be resuspended in 50-200 ml of TE. Allow the DNA to dissolve overnight at 4íC.
This DNA is now ready to be used in PCR or southern blot analysis.