1. Put 1 cm tail in 1.5 ml microcentrifuge tube.
2. Add 500µl Tail Buffer and 30 µl Proteinase K. (see recipes below).
3. Incubate O/N at 55°ree;C (or 4 hours minimum).
4. Mix well by shaking by repeated inversion. Do NOT vortex.
5. Let cool to room temperature.
6. Pre-Spin (to get rid of fur) 8 min (usually 4°ree;, TRm ok).
7. Pour supernatant into a clean tube (numbered). (Toss out furry tube).
8. Add 250 µl saturated NaCl (~6M) and invert to mix (don't vortex) to precipitate the proteins.
9. Let sit for 5 minutes at room temperature.
10. Centrifuge max speed in microfuge (~13000 rpm), 10 min at 4°ree;C, to spin down the protein. (TRm is ok, but 4°ree; is much better).
11. Pipet supernatant into a fresh tube. Avoid transferring the white precipitate.
12. Add 500µl isopropanol.
13. Mix by inversion until a white thready material is visible (DNA!). Shake until thread doesn't get any bigger, ~ a minute.
14. Centrifuge max speed in microfuge (~13000 rpm), 10 min (TRm is ok).
15. Pipet off the sup, being careful not to disturb the pellet (DNA).
16. Wash pellet with 70% EtOH (100-800 µl). Vortex briefly.
17. Centrifuge 6 minutes.
18. Pipet off the sup, being careful not to disturb the pellet (DNA).
19. Tap/Drain tube onto a kimwipe.
20. Air Dry for 45-60 min. (Do NOT use Speed-Vac).
21. Dissove the pellet in 100µl Tris (10mM Tris, or useTE) containing RNase.
22. Let dissolve O/N at 4°ree;C. IMPORTANT- DON'T SKIP THIS STEP!
Mix well. Measure OD260 or OD260/280.
Use 20 µl for Southerns, or check [DNA] and use 10mg.
Tail Prep Solutions(non-phenol prep.)
RNase: 10 mg/ml. Use 20µ RNase per 1 ml Tris.
Proteinase K (Sigma #63173): 10 mg/ml in H2O, (activate 1 hr at 37°ree;C before use).
Saturated NaCl: Add NaCl to approximately 6M (35g/100ml).
| Stock | Reagent | Vol. For 100 mls | Final |
|---|---|---|---|
| 1M | Tris | 5 mls | 50 mM |
| 0.5M | EDTA | 20 mls | 100 mM |
| 10% | SDS | 5 mls | 0.5% |
| mQ H2O | 70 mls | ||
Shirley Reynolds