Preparing mouse tail DNA for genotyping PCRs:

Materials needed:

Tail digestion buffer (recipe below)

Proteinase K solution (Roche 3115828; conc. ~ 20 mg/ml, or effectively 200x)
Ear punch, scissors, forceps (kept in mouse room)
PCR strip tubes and caps (label ahead of time and bring from lab)
Isopropanol
70% ethanol
TE

Note: To avoid contamination of genomic DNA preps with plasmid or other DNA, use great care in
pipetting. When going into stock buffers or stock solutions, use filter tips and disposable plastic
pipets, and set aside a separate bottle of TE for genomic DNA preps.
1.

Tag and Sample: Ear-punch young mice (17-21 days of age) to number (start at #1 for each
new litter) and cut off small (2-5 mm) sample of tail tip. Transfer tail sample to corresponding
PCR tube. Cap PCR strips, return to lab.

2.

Spin or tap PCR strip tubes to knock tail tips to bottom of tubes, open carefully (avoid having
tail tips pop out and get mixed up!).

3.

Digest: Assuming 150 uL per tail sample, prepare appropriate volume of digestion solution
[digestion buffer (see below) + 0.1 mg/ml (1:200) proteinase K].

4.
5.

Add 200 uL digestion solution per tube. Cap tubes tightly.

Transfer tubes to hybridization bottle, incubate on hybridization oven rotisserie overnight at
55C. If hyb oven is not available, set benchtop incubator to 55C -- in the morning, shake tubes to
disperse gunk, and return to oven for extra 1-3 hrs.

6.

Remove tubes, let cool. (Digests can be frozen at -20C at this stage, if desired.)

7.

Spin down at top speed, 1 min, to pellet undigested hair/debris.

8.

Precipitate: Label a second set of PCR strip tubes with appropriate numbers (use alcoholresistant marker, so that the labels dont come off if you splash ethanol while washing), add 100
uL isopropanol to each (a repeating pipettor is useful). Transfer 100 uL digestion supernatant
(using multi-channel pipettor) to the PCR strip tubes containing isopropanol, cap tightly.

9.

Place in tube rack and shake up and down (put another tube rack on top when you shake, to
keep the strips from flying away!) -- genomic DNA should form visible stringy precipitate. (Tail
preps can be stored indefinitely at this stage, at 4C.)

10.

Spin down at top speed, 2 min.

11.

Rinse: Invert strip tubes on a paper towel (pellet should be tightly adherent), rinse with approx.
200 uL 70% EtOH (a repeating pipettor is useful, but a squeeze bottle is fine as well).

12.

Spin down and dump supernatant as above. Blot on paper towel ~ 5 min, then return to rack
and let air-dry for ~ 5 min more.

13.

Rehydrate: Add 200 uL TE to each sample (repeating pipettor can be used, with care). To
avoid contamination, be absolutely sure not to return pipet tip to TE bottle after it has come in
contact with a sample tube.

14.

Cap tubes, incubate overnight at 55C, or 2-3 hrs at 55C to dissolve DNA, rotating in
hybridization oven.

15.

Vortex to mix contents, let cool. Spin down at top speed, 1 min to pellet any residual gunk
before using for PCR (50 l per reaction.) Can be stored for months at 4C.

Digestion buffer
final)
0.1 M Tris pH 8.5
5 mM EDTA
0.2 M NaCl
0.2 % SDS

stock conc.

volume

volume (500 ml

5 ml
50 ml
0.5 ml
5 ml
2 ml
20 ml
1 ml
10 ml
50 ml final (with dH2O)
500 ml final (with dH2O)
- SDS will initially crash out of solution when added; bring up to final volume and shake to mix.
- A working stock should be kept in a 50 ml conical; avoid going in and out of a large stock bottle,
to minimize risk of contamination.

------Charlie Murtaugh
Charlie Murtaugh
Charlie Murtaugh
Charlie Murtaugh
Charlie Murtaugh
Jeff Stagg

1M
0.5 M
5M
10%

(50 ml final)

4/25/05
6/21/05
2/11/06
8/18/08
3/6/12
9/24/14