1. Remove 0.5 cm of tail into polypropylene microfuge tube (do not mince).

(The tubes must have tight-fitting caps, so that there are no leaks in steps 3 and 7 below.)

2. Add 0.5 ml DNA digestion buffer with proteinase K added to 0.5 mg/ml final concentration.

(0.5 mg/ml is a high concentration and can probably be reduced.)

DNA digestion buffer: 50 mM Tris-HCl pH 8.0       100 mM EDTA pH 8.0       100 mM NaCl      1% SDS 3. Incubate overnight at 50-55 °C with gentle shaking.

(At this step, mechanical agitation greatly aids complete disruption of the tail.)

4. Quick spin tubes to get solution off inside of cap.

5. Fill inside well of microfuge tube cap with vacuum grease.

(We use Dow Corning high vacuum grease and a 10cc syringe to dispense.)

6. Add 0.7 ml neutralized phenol/chloroform/isoamyl alcohol (25:24:1).

7. Mix fairly vigorously. (Do NOT vortex--We use a clinical rotator for 1 hour.)

8. Spin in microfuge at top speed 5 minutes and transfer 0.5 ml of the upper phase to new microfuge tube.

(Use P1000 for transfer, and draw the aqueous phase gently through tip several times after transfer if the DNA is still in large, gelatinous mass.)

9. Add 1 ml 100% ethanol at room temperature and invert (using clinical rotator if you wish) until DNA precipitate forms.

(approximately 1 minute)

10. Spin in microfuge 5 minutes and carefully remove and discard supernatant.

11. Add 0.5-1 ml 70% ethanol (-20 °C) and invert several times.

12. Spin in microfuge 5 minutes and carefully remove and discard supernatant.

13. Quick spin tubes and remove last drop of ethanol solution with 25 µl capillary tube.

14. Air dry at room temperature or in dessicator (overnight if you wish).

15. Add 100-200 µl TE buffer and incubate at 65 °C for 15 minutes to resuspend DNA. Draw DNA through P1000 tip after 65 °C incubation to aid in suspension if you wish.

16. Use 10-20 µl for restriction enzyme digest.

17. Total yield is approximately 20-50 µg DNA, 0.1-0.25 µg/µl.