June 28, 2016

Tail DNA Preparation


(M.Fero 10/2013)


Lysis Buffer: (10 mM Tris pH8, 100 mM NaCl, 25 mM EDTA, 0.5%SDS), store at room temp.

10 mg/mL Proteinase K: 100 mg (Roche) + 10 mL buffer (10 mM Tris pH8.0, 20 mM CaCl2, 50% (v/v) glycerol), store at -20ºC in 1.5 mL aliquots.

Lysis buffer + proteinase K (1 mg/mL): 50 mL Lysis buffer (above) + 0.5 mL 10 mg/mL proteinase K, store at -20ºC.

TE: 10 mM Tris pH8, 1mM EDTA

Phenol: Molecular biology grade. Equillibrate 1x with Tris pH8, and then 1x with TE Store at 4°C.

Chloroform: Fresh choloroform + 1/20 vol. isoamyl alcohol. Store at r.t.
Absolute ethanol.


  1. Cut 3 mm of toe or tail or toe tips from 7-10 day old mice into 1.5 mL microcentrifuge tubes.
  2. Digest tail biopsy by adding 0.7 mL lysis buffer + proteinase K and incubate for 4-16 hrs at 37°C.
  3. Add 0.7 mL of phenol (or a 1:1 (v:v) mix of chloroform:phenol) and slowly rotate at 4°C for 1 hr. to overnight.
  4. Spin at high speed (15,000 g) for 2 minutes to separate phases. The hair and other debris should pellet to the bottom. Pipet the top (aqueous) phase to a fresh tube, taking care to avoid the bottom (organic) layer.
  5. Repeat the extraction (as in steps 2-3) with 0.7 mL of phenol or a 1:1 (v:v) mix of chloroform:phenol for 1 hr.
  6. Repeat the extraction (as in steps 2) but with 100% chloroform. You should now have ~0.7 mL of aqueous solution in each tube. Spin for 2 min. Transfer 0.5 mL the solution to a fresh tube, taking care not to place the pipet tip to the bottom of the tube (since this is where any residual CHCl3 will be located).
  7. Precipitate by adding 2 volumes (1.0 mL) of absolute ethanol and mix vigorously. You should see some DNA stranding at this point. Incubate at -20°C for >30 min. Samples may be stored in -20ºC in ethanol indefinitely.
  8. Spin at 12,000 rpm for 5 min. Discard supernatant. Wash pellet by adding 1 mL of 70% ethanol, mix by inversion, and spin for 1 minute. Discard supernatant being taking care not to pour out the pellet. It is likely to be loosely adherent since the salt is washed out. Repeat the 70% ethanol wash, spin and again discard the supernatant. Blot the excess ethanol carefully on a clean paper towel. Invert the tube on a paper towel to air dry the tube.
  9. Resuspend pellet in 0.1-0.5 mL TE (depending on the amount of DNA). Use 1 µL for a PCR reaction, or 1/2 of the sample for a Southern blot. Store the genomic DNA at 4ºC for 1-2 weeks, or at -20ºC for longer periods.

Quality Control - Options for QC include the following:

  1. Run 2 µL of the genomic DNA on a 0.6% agarose gel, with high MW marker, e.g. lambda HindIII. The DNA should form a high MW band (20-50 kb), with minimal smearing or degradation below this level. RNA may also be present if a metabolically active source tissue was used instead of toe/tail tip. Insoluable material, or protein contamination, may result in ethidium bromide staining that does not migrate out of the loading well.
  2. A spectrophotometer (e.g.Nanodrop) may also be used to measure the A250/A280 ratio, as a crude indicator of DNA purity. Values of 1.9-2.0 are expected. Values > 2.0 may indicate organic solvent contamination; values <1.9 represent protein contammination.

Quantitation - Options for quantifying genomic DNA include:

  1. The DNA concentration may be estimated by comparing band intensity on an agarose gel to lamda HindIII or other standards with known band quantitities.
  2. The spectrophotometric A260 value may be used to estimate DNA concentration, if the DNA is high quality. ([DNA] mg/mL = A260 x 50)
  3. Fluorometry with a DNA binding dye (SybrGreen or Höchst) is an accurate and sensitive measure of DNA concentration.

Tags: Fero-Lab-Protocols, Fero-Mice