Tail DNA Purification Protocol

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Tail DNA Purification Protocol

 

Protocol for isolating High Molecular Weight DNA from mouse tails


(from Hogan et al., Manipulating the Mouse Embryo, Cold Spring Harbor, 1986, 1994)

 

Day 1

1. Cut 0.5 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 0.7 ml of 50 mM Tris (pH 8.0), 100 mM EDTA, 0.5% SDS to each tube. Add 35 ul of a fresh 10 mg/ml solution of Proteinase K dissolved in 50 mM Tris (pH 8.0), 1 mM CaCl2.

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

 

Day 2

 

4. Remove tubes from 55°C. Add 0.7 ml phenol (equilibrated with Tris pH 8.0). Close tube and shake vigorously for 3 min, so that phases mix completely.

5. Centrifuge 3 min in a microfuge. Phases will separate.

6. Transfer upper aqueous phase to a fresh tube, being careful not to pick up phenol or material at the interface.

(note, We have had good results using Phase Lock Gel I tubes at this step (Catalog #p1-188233, from 5'-3', Inc., but it is not required.)

7. Add 0.7 ml of phenol/chloroform (1:1), shake vigorously for 2 min, and centrifuge for 2 min.

8. Again remove aqueous phase, avoiding interface, and transfer to a fresh tube (1.5 ml).

9. Add 70 ul 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 and should not be used.

10. Spin in a microfuge for 30 seconds to pellet DNA. Remove and discard as much ethanol supernatant as possible.

11. Add 1 ml of 70% ethanol (room temperature) to tube, and vortex or shake vigorously to wash the DNA pellet. This step is essential to remove traces of SDS and phenol.

12. Microfuge for 1 min at room temperature. Remove as much ethanol as possible. Dry DNA briefly in vacuo.

13. Resuspend the DNA pellet in 0.1 ml 10 mM Tris, pH 8.0/1 mM EDTA. Leave at room temperature several hours or heat at 65°C for 5-10 min to dissolve completely. The DNA should have an A260/A280 of >1.7. The concentration should be calculated using 50 ug/ml = 1.0 A260. Use 10 ug of each DNA sample for Southern blot analysis.

14. DNA prepared in this manner will contain a substantial amount of RNA, but this will not interfere with restriction digestion or Southern blot analysis. 5 mg of DNAse-free RNaseA can be added to each sample during restriction digestion if you like.

15. Be sure to include appropriate positive and negative controls with your Southern analysis. A negative control should be normal mouse DNA, usually 1 ug for PCR or 10 ug for Southern blot. Positive controls should be addition of your transgene to normal mouse DNA, at one, five and ten single copy equivalents of the fragment. (This will usually be in the range of 1-4 pg of plasmid DNA for single copy. See page for calculation of copy standards.) Examples can be found on page 9 of our manual, or the transgene detection web page.