Posted on May 16th, 2016 by UNM CC
- 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.
Lysis buffer + 1 mg/mL proteinase K: 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 T.E. Store at 4°C.
- Chloroform: Fresh choloroform + 1/20 vol. isoamyl alcohol.
- Absolute ethanol.
- Cut 3 mm of toe or tail or toe tips from 7-10 day old mice into 1.5 mL microcentrifuge tubes.
- Digest tail biopsy by adding 0.7 mL lysis buffer + proteinase K and incubate for 4-16 hrs at 37°C.
- Add 0.7 mL Phenol and slowly rotate at 4°C for 1 hr. - overnight.
- 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. Add 0.7 mL of a 1:1 (v:v) mix of chloroform and and phenol and rotate at 4°C for 1 hr.
- Repeat step 3 with 100% chloroform.
- Spin for 2 min, and transfer 0.5 mL aqueous phase to a fresh tube. Be careful not to transfer any chloroform at this step. Precipitate by adding 2 volumes (1 mL) of absolute ethanol and mix vigorously. Incubate at -20°C for at least 30 min. Samples may be stored in -20ºC in ethanol indefinitely.
- Spin at 12,000 rpm for 5 min. Discard supernatant. Wash pellet by adding 1 mL of 70% ethanol, gently invert, and spin for 1 minute. Discard supernatant being taking care not to pour out the pellet which is likely to be loose adherent at this point. 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.
- Resuspend pellet in 0.5 mL TE Use 1 µL for a PCR reaction, 0.25 mL for a Southern blot.