Protocol for Isolating High-Molecular-Weight DNA from Mouse Tails
(From Manipulating The Mouse Embryo; Hogan et al. Cold Spring Harbor, 1986)
- Cut 0.5-1.0 cm of tail and place in a 1.5-ml microfuge tube (tails can be frozen at -20°C prior to extraction). These tail biopsies will be performed by the Core.
- Add to the tube 0.7 ml of 50 mM Tris (pH 8.0), 100 mM EDTA, 0.5% SDS. Add 35 ul of a fresh 10 mg/ml solution of Proteinase K.
- Incubate at 55°C overnight on a gently rocking platform.
- Next morning, remove tubes from 55°C. Add 0.7 ml of phenol (equilibrated with Tris - pH 8.0). Close tube and shake vigorously for 3 minutes., so that phases mix completely.
- Centrifuge in a microfuge for 3 minutes. Phases will separate.
- Transfer upper aqueous phase to a fresh tube, being careful not to pick up phenol or material at interface.
- Add 0.7 ml of phenol/chloroform (1:1), shake vigorously for 2 minutes and centrifuge for 2 minutes.
- Again remove aqueous phase, avoiding interface, and transfer to a 1.5-ml tube.
- Add 70 ml of 3 M sodium acetate / pH 6.0 (i.e., 1/10 volume), and 0.7 ml of 100% ethanol at room temperature. Shake to mix thoroughly. DNA should immediately form a stringy precipitate. Sodium acetate with a pH lower than 6.0 will cause the EDTA to precipitate.
- Spin in microfuge for 30 seconds to pellet DNA. Remove and discard as much ethanol supernatant as possible.
- Add 1 ml of 70% ethanol (room temperature) to tube, and vortex or shake vigorously to wash DNA. This step is essential to remove traces of SDS and phenol.
- Centrifuge in microfuge for 1 minute at room temperature. Remove as much ethanol supernatant as possible. Dry DNA briefly in vacuo.
- Add 0.1 ml of 10 mM Tris (pH 8.0)/1 mM EDTA to tube. Leave at room temperature overnight to dissolve. If necessary, DNA can be dissolved more quickly by heating for 5-10 minutes at 65oC. The DNA should have an A260/A280 of > 1.7, and the concentration should be calculated using: 50 ug/ml = 1.0 OD260. Use 10 ug of each DNA preparation for Southern blot analysis.
- DNA prepared in this manner will contain substantial amounts of RNA, but this does not interfere with restriction enzyme digestion or Southern blot analysis. When performing restriction enzyme digestion, add 5 ug of DNase-free RNase A to each sample along with the restriction enzyme. Digestion with certain restriction enzymes may also be aided by adding 4 mM spermidine to the reaction.
- Make sure you include the appropriate positive and negative hybridization controls for your Southern analysis.