Transgenic Mouse Facility

Isolating DNA from mouse tail clips

Note: Tail clips will be approximately 100 mm long.

Procedure

  1. Label microfuge tubes with mouse ear tag number.
    Put tail clips in 0.7 ml of Lysis buffer (recipe below) + proteinase K (dilute solution to 1mg/ml in Lysis buffer).
    Make sure tops are tightly closed.
    Rotate or shake overnight at 50°C.
  2. Next morning, label one set of microfuge tubes with mouse ear tag numbers.
  3. Vortex the tail digests.
    Spin samples in microfuge for 10 minutes at maximum speed.
  4. Hair and undigested debris may be in the precipitate.
    The supernatant may be colored but this does not effect the DNA isolation.
    Carefully transfer the supernatant (maybe thick and hard to pipette---use wide bore tips) to a clean tube with identical number.
    Add an equal volume (0.7 ml) of isopropanol (2-propane) to supernatant.
  5. Mix by inversion several times until white strands of DNA become visible.
  6. Spin the tubes at 10,000 x g for 5 minutes.
    Remove supernatant being careful not to disturb the DNA pellet in the bottom of the tube.
  7. Wash each DNA pellet with 0.5 ml of ice-cold 70% ETOH.
    Centrifuge and carefully remove all the ETOH wash (use a yellow pipette tip if necessary).
    Turn tubes upside down on paper towel to air-dry the DNA (do not over-dry or the DNA may be difficult to resuspend).
  8. When DNA pellets are dry (i.e., all traces of ETOH are gone), resuspend the DNA in 0.5 ml genomic DNA buffer (recipe below).
    Cap securely and rotate or shake DNA overnight at 37°C overnight to ensure complete DNA solution.
  9. Next morning, read A260, A260/280 absorbance (usually takes a 1:100 dilution for accurate OD).
    Calculate DNA concentration and proceed to screening step(s) such as Southern blot or PCR assay.

Recipes

Lysis buffer

  • 50 mM Tris, pH 8.0
  • 100 mM EDTA
  • 100mM NaCl
  • 1% SDS

Genomic DNA buffer

  • 10 mM Tris, pH 8.0
  • 0.1 mM EDTA

Instructions

Make both solutions in H20 and filter sterile with 0.2µm Nalgene filter bottle.

If SDS precipitates warm in 37°C water to re-dissolve.

Proteinase K stock is 10mg/ml in sterile water.

  • Freeze at -20°C; thaw as needed. (Source Roche cat# 1092-766)

Reference

PW, Laird et al. “Simplified Mammalian DNA Isolation Procedure”, NAR 19: 4293, 1991.