Tail Preps: DNA Isolation From

NOTE: THIS IS FINE FOR SOUTHERNS, BUT NOT FOR SCREENING BY PCR.

(from Ruixia, 7/99, from protocol by Stef Oehen 7/94).

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℃ (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℃, 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℃, to spin down the protein. (TRm is ok, but 4℃ 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℃. 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℃ before use).

Saturated NaCl: Add NaCl to approximately 6M (35g/100ml).

Tail Buffer

Stock Reagent Vol. For 100 mls Final

Shirley Reynolds