John M. S. Bartlett.pdf - Bio-Nica.info

58 Schmerer
2. Materials
To apply the protocol described in the next section, the following chemicals and
solutions are required.
1. 0.5 M EDTA solution (pH 8.3).
2. Sterile water (Ampuwa ® , Fresenius).
3. Proteinase K-solution (approx 2 g/mL, e.g. Perkin–Elmer).
4. 70% phenol/chloroform/water (24251, e.g., Rotipuran, Roth, or Perkin–Elmer) or,
alternatively, a 5 M solution of sodium perchlorate in sterile water.
5. Chloroform (100%, e.g., Perkin–Elmer).
6. 2 M sodium acetate buffer (pH 4.5, Perkin–Elmer).
7. Isopropanol (abs., Merck).
8. Silica solution (Glasmilk , Dianova).
9. Ethanol (abs.).
3. Methods
1. To prevent coprocessing of possible adhering contaminations, exposed surfaces of the
bone sample are removed by the use of a scalpel. Subsequently, the material is exposed to
ultraviolet light for 15 min on each of the surfaces.
2. According to the consistency of the material, samples are ground to a fine powder using a
mixer mill (MM2, Retsch) or a mortar and pestle.
3. Bone powder (0.3 g) is mixed with 1.5 mL of 0.5 M EDTA-solution (pH 8.3) in a 2-mL
reaction tube (Eppendorf), vortexed vigorously, and incubated for 96 h in a shaking
waterbath at 20°C and constant shaking (for decalcification of the bone material).
4. Residues of the bone powder are pelleted by centrifugation at 3000g for 5 min in a 5415 C
bench-top centrifuge (Eppendorf) or equivalent. The following steps are performed with the
supernatants (approx 1.3 mL). These are transferred to an automated DNA extraction system
(Gene Pure, Perkin–Elmer) or alternatively to a 15-mL tube (e.g., BlueMax , Falcon).
5. The aqueous supernatants are mixed with 1.3 to 1.8 mL sterile water and incubated with
380 to 650 µL proteinase K-solution (approx 2 g/mL) at 60°C for 1.5 h.
6. Two volumes (2× supernatant volume) of 70% phenol/chloroform/water (24251) are
added to the solution. The suspension is constantly shaken for 6 min at room temperature.
Alternatively, phenol can be replaced by 2 mL of a 5 M sodium perchlorate-solution (ref. 13,
see Notes 2 and 3).
7. Using phenol—to facilitate the process—phase separation is performed at 60°C for 8 min
and the phenolic layer is removed. Alternatively, the suspension can be separated by
centrifugation at 4500g for 10 min. Using sodium perchlorate, no phase separation occurs.
After incubation at 60°C for 8 min, the extraction mix is therefore processed according
to step 8.
8. The aqueous phase is mixed with 4.0 to 5.3 mL of chloroform (100%) and the resulting
suspension shaken for 6 min at room temperature.
9. Phase separation is performed at 60°C for 8 min and the chloroform phase is subsequently
removed. Alternatively, the suspension can be separated by centrifugation at 4500g for
10 min.
10. DNA precipitation takes place in the presence of 64 to 120 µL of 2 M sodium acetate-buffer
(pH 4.5) and 2.8 to 3.8 mL of isopropanol (abs.) at room temperature. After mixing these
components for 1 min, the pH of the extraction mix should be determined. If necessary,
the pH value should be adjusted to a maximum of 7.5 by further addition of sodium
acetate buffer to ensure optimal precipitation of DNA (see Note 4). Subsequently 5 µL