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Transgenic & Gene-targeting Institutional Facility
Transgenic & Gene-targeting Institutional Facility  :  Protocol  :  Isolation of DNA form Mouse Tails

Isolation of DNA form Mouse Tails

  • Day 1

    1. Cut 1.0 cm of tail and place into a 1.5ml microcentrifuge, place on ice.  Add to the tube 0.62ml of Digestion Buffer (100mM NaCl, 50mM Tris-HCl, pH 8.0, 100mM EDTA, pH 8.0, 1% SDS) and 30.0ul of protease K solution (25 mg/ml).
    2. Incubate at 55°C overnight on a rocking platform or in a water bath, mixing occasionally.

    Day 2

    1. Cool the solution to room temperature.  Add 10ul of 1 mg/ml RNase A (DNase free) and incubate at 37°C for 1 hour, gently mixing intermittently.  Extract the aqueous supernatant once with buffered phenol (1 volume) and twice with phenol: chloroform: isoamyl alcohol (24:25:1 volume) shaking vigorously for 30 seconds and centrifuging at 12,000 rpm for 3 minutes for each extraction.  Remove final aqueous phase and place into a fresh microcentrifuge tube.
    2. Add an equal volume of isopropanol at room temperature and invert the tube several times until a precipitate forms.  Centrifuge 12,000 rpm for 30 seconds to pellet DNA, discard supernatant.
    3. Add 1.0ml of 70% ethanol (room temperature) to rinse pellet, centrifuge 12,000 rpm for 1 minute.  Discard ethanol and air-dry DNA pellet.
    4. Add 100-200ul of ddH2O or TE buffer (pH 8) to tube.  Incubate tubes at 55°C until DNA is dissolved.
    5. Measure the DNA concentration at 260 nm and 280 nm (OD260/280>1.75).  Use 2-10ug DNA for a Southern blot analysis or 100ng of DNA for a PCR reaction.
    6. Store the DNA at 4°C for up to 6 months.

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