Protocols

Protocol for Isolating High-Molecular-Weight DNA from Mouse Tails

(From Manipulating The Mouse Embryo; Hogan et al. Cold Spring Harbor, 1986)

Day 1

  1. 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.
  2. 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.
  3. Incubate at 55°C overnight on a gently rocking platform.

Day 2

  1. 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.
  2. Centrifuge in a microfuge for 3 minutes. Phases will separate.
  3. Transfer upper aqueous phase to a fresh tube, being careful not to pick up phenol or material at interface.
  4. Add 0.7 ml of phenol/chloroform (1:1), shake vigorously for 2 minutes and centrifuge for 2 minutes.
  5. Again remove aqueous phase, avoiding interface, and transfer to a 1.5-ml tube.
  6. 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.
  7. Spin in microfuge for 30 seconds to pellet DNA. Remove and discard as much ethanol supernatant as possible.
  8. 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.
  9. Centrifuge in microfuge for 1 minute at room temperature. Remove as much ethanol supernatant as possible. Dry DNA briefly in vacuo.
  10. 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.
  11. 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.
  12. Make sure you include the appropriate positive and negative hybridization controls for your Southern analysis.