Biomedical Engineering Reference
In-Depth Information
Proteinase-K (10 mg/ml)
•
PPS: protein precipitation solution (e.g. Promega, A7951)
•
TE: 10 m
M
Tris-HCl, pH 8.0, 0.1 m
M
EDTA
•
Tissue arrayer (e.g. Beecher Instruments)
•
Method
1 Identify histologically normal and tumor areas from the FFPE tumor. Using a tissue
arrayer, collect three punches (0.6 mm) from normal tissue and three from tumor tissue
in separate tubes.
2 Add 1 ml of xylene to a tube with three FFPE tissue punches.
3 Vortex and mix on a rotating wheel for 15 min at room temperature.
4 Centrifuge at 13 000
g
for 3 min at room temperature.
5 Carefully remove the xylene from the tissue cores.
6 Repeat steps 2-5.
7 Add 1 ml of 100% (v/v) ethanol.
8 Vortex and mix on a rotating wheel for 15 min at room temperature.
9 Centrifuge at 13 000g for 3 min at room temperature.
10 Carefully remove ethanol from the tissue pellet.
11 Repeat steps 7-10.
12 Air dry for 5 min.
13 Add 150
μ
l of PK1 buffer to the dried tissue pellet.
14 Add 5
μ
l of proteinase-K.
15 Mix and pulse centrifuge
16 Incubate overnight in a heat block at 56
◦
C.
17 Heat-inactivate the proteinase-K at 100
◦
C for 10 min.
18 Centrifuge at 13 000
g
for 10 min at room temperature.
19 Transfer the supernatant containing the DNA to new tube.
20 Add 80
μ
l of NucleoSpin buffer B3.
21 Vortex twice for 5 s each, and incubate at 70
◦
C for 5 min. Vortex briefly at the end of
the incubation.
22 Allow the lysate to cool down to ambient temperature.
23 Add 80
μ
l of 100% (v/v) ethanol to the lysate.
