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)
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.
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.