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        Tail Preps: DNA Isolation From Mouse Tails Without Phenol

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        <center> <h3> <font>Tail Preps: DNA Isolation From Mouse Tails Without Phenol</font></h3> </center>

        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°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).

         

        <center> <h3> <font><font>Tail Buffer</font> </font></h3> </center> <center>  </center>
        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    

         

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