- Tail buffer 50 mL
- Phenol/chloroform (1:1 mixture)
- 0.5M EDTA
- 4M NH4Ac
- Absolute EtOH
- 70% EtOH
- Tris/EDTA pH 7.5
- Proteinase K (20 mg/mL dissolved in distilled H2O)
- This works best with mice that are at weaning age. Take 1 toe or 1 mm
of tail and place it in a ependorf tube. Don’t take more than this, it isn’t
necessary, and too much material interferes further down the track. Also,
if the amounts taken are consistant between samples, then the amounts
used for the Southern will be even between samples. If there are a number
of samples to be collected, place tubes on ice.
- Make up a mix of tail buffer and Proteinase K, allowing 600 mL of tail
buffer with 500 mg/mL Proteinase K for each sample (plus an extra dose
in case of inaccurate pipetting). Place in a waterbath at 55°;C all day, or
at least 2 hrs.
- Samples are transferred to hot air shaker overnight at 62°;C at a mediumshake
- To each eppendorf, add 500 mL of phenol/chloroform (lower phase of mix).
Mix by inversion/shake (don’t vortex as this shears the DNA). Spin at
13000 rpm for 2 minutes. Collect supernatant, being careful to avoid the
- Place supernatant in a fresh eppendorf tube and add:
- 2 µL 0.5M EDTA pH 8.0
- 200 µL 4M NH4Ac
- 800 µL isopropanol
- Mix by inversion/shake, Spin at 13000 rpm for 5 minutes. Remove and
discard supernatant, being extremely careful not to disturb the pellet.
- To the tube with the pellet, add 200 µL 70% EtOH and vortex. Spin at
13000 rpm for 2 minutes. Remove and discard supernatant (watch the
- Add 200 mL TE and vortex, allowing DNA to resuspend at room temperature.
- Once DNA has resuspendend, digests may be set up. 30 mL of
DNA/TE suspension per digest should be sufficient, RNAse is not necessary.
Digest overnight, precipitate with 12 mL 5 m NaCl, 600 mL EtOH and
resuspend pellet in 18 µL TE and 7 µL dye. Allow to dissolve for 20 min
RT and heat to 37°;C for 5–10 min before loading.