Tech Tips tenplate temp 10/12/98 5:50 pm Page 3

Technical tips

Isolation of DNA from cryostat sections

of bone using Nucleon BACC 1
B. Noble
Bone Research Group, Cambridge University Dept. of Med.,
Addenbrookes Hospital, Cambridge, CB2 2QQ, UK

Osteocytes undergo apoptotic cell death under certain circum-

stances which may contribute to the control of bone modelling
and re-modelling (1). One characteristic used in the study of
apoptosis is the fragmentation of genomic DNA into oligo-
nucleosomal sized increments which result in a classic DNA
ladder when separated on an agarose gel.

Extraction of DNA from mineralized bone tissue is problem-

atic. A modification of the Nucleon™ BACC 1 protocol has
therefore been developed enabling DNA to be extracted from
frozen bone material which has been sectioned using a cryostat.

Procedure
1. Coat freshly isolated bone in 5% PVA and snap freeze in
a hexane chilling bath. Store at -70 °C.

2. Cut 20 undecalcified sections per sample (10 µm thick)

using fine forceps to remove only the bone component
of each section, leaving behind marrow and surrounding
tissue. Remove bone material to a chilled pot to be
stored at -70 °C prior to use.

3. Immerse sections in 340 µl Reagent B for 40 min before

spinning briefly at ~600 g to soft pellet any debris.

4. Recover supernatant and add 100 µl sodium perchlorate.

Incubate at 37 °C for 20 min then 65 °C for 20 min. Add Figure 1. Schematic diagram for the isolation of DNA from bone.
580 µl chloroform and mix at room temp for 20 min.
bp op calv
5. Transfer to Nucleon insert tubes, centrifuge, then add
45 µl Nucleon resin and centrifuge again.
bp
6. Recover the aqueous phase and precipitate with 2
volumes ethanol (~880 µl). Pellet the DNA and wash
2,072 –
with 70% ethanol. (Treat the first ethanol supernatant,
1,500 –
which potentially contains DNA fragments, with 0.2 M
sodium acetate and leave at -20 °C for 24 hr and pellet
the DNA).
600 –
7. Air dry the pellets at room temp. Add ~20 µl TBE to
each sample and incubate at 65 °C for 3 min. Add
loading buffer after cooling.

8. Samples are run on a 1.5% agarose gel

The procedure is shown schematically in Figure 1. Typical 100 –

results are shown in Figure 2.

Reference Figure 2. Gel photo showing the classic

1. Noble, B. S. et al., Bone 20, 273-282 (1997). ladder pattern of apoptotic DNA (arrows).
bp = 100 base-pair marker
op = osteophyte DNA
calv = skull DNA

Life Science News 1, 1998 Amersham Biosciences