Protocol for isolating High Molecular Weight DNA from yolk sacs of 9.5 dpc or later embryos

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

Day 1

1. Dissect embryos and place each yolk sac in a 1.5 ml microfuge tube (can be frozen at -20°C prior to extraction).

2. Add 30 µl of 50 mM Tris (pH 8.0), 100 mM EDTA, 0.5% SDS to each tube (use 50-100 µl for 10.5 dpc or later embryos). Add 1.6 ml of a fresh 10 mg/ml solution of Proteinase K dissolved in 50 mM Tris (pH 8.0), 1 mM CaCl2 (final conc of 500 µg/ml).

3. Incubate at 50°C several hours to overnight; agitation is not required.

Day 2

4. Remove tubes from 50°C. Add 5-10 µg of carrier tRNA to aid in later recovery (1 µl of 10 mg/ml per sample).

5. Add 32 µl phenol (equilibrated with Tris pH 8.0). Close tube and shake vigorously for 3 min, so that phases mix completely (do not vortex).

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

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

8. Add 32 µl of phenol/chloroform (1:1), shake vigorously for 2 min, and centrifuge for 2 min.

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

10. Add 32 µl of chloroform:isoamyl alcohol (24:1), shake for 2 min, and centrifuge for 2 min.

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

12. Add 3 µl of 3 M sodium acetate, pH 6.0 (i.e. 1/10 volume), and 33 µl 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.

13. Spin in a microfuge for several minutes to pellet DNA. Remove and discard as much ethanol supernatant as possible.

14. Add 300 µl 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.

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

16. Resuspend the DNA pellet in 30 µl 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 µg/ml = 1.0 O.D260. Expect <5 µg from a 9.5 day yolk sac, 25 µg from a 11.5 d yolk sac and 50-100 µg from a 13.5 d yolk sac. Use 10 or 20 µg of each DNA sample for Southern blot analysis.

17. 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 µg of DNAse-free RNaseA can be added to each sample during restriction digestion if you like.