Biomedical Engineering Reference
In-Depth Information
PROTOCOL 3.1 Non-Column-Based DNA Isolation
from FFPE Tissue
a
Equipment and reagents
Xylene
•
Ethanol (100% (v/v), 70% (v/v))
•
PK1: 10 m
M
Tris-HCl, pH 8.3, 50 m
M
KCl, 2.5 m
M
MgCl
2
, 0.45% (v/v) NP-40, 0.45% (v/v)
Tween-20, 0.01% gelatin.
•
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)
•
b
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.
