Biomedical Engineering Reference
In-Depth Information
19 Transfer the supernatant containing the DNA to new tube.
20 Add 50
μ
l of PPS to the supernatant.
21 Vortex and chill on ice for 5 min.
22
c
Centrifuge at 13 000
g
for 5 min at room temperature.
23 Carefully transfer the supernatant containing the DNA to a new tube.
24
d
Add 150
μ
l of isopropanol (at room temperature) to the supernatant and mix by
inverting the tube.
25 Precipitate the DNA by centrifuging at 13 000
g
for 5 min at room temperature.
26 Carefully remove the supernatant.
27
e
Wash the pellet with 200
μ
l of 70% (v/v) ethanol.
28 Repeat steps 26 and 27.
29 Air dry the DNA
f
pellet and dissolve in 100
μ
lofTE.
Notes
a
FortheGoldengateassay,wefavortheuseofDNAisolated using a relative simple precipitation
procedure after proteinase K digestion. Although column, or bead based methods, are likely to
yield cleaner DNA, smaller fragments from the already fragmented FFPE tissue DNA are better
preserved using a precipitation method.
b
Genomic DNA can be isolated from fresh frozen tumors or from normal blood leukocytes, using
the Wizard Genomic DNA Purification Kit (Promega).
c
The precipitated protein will form a white pellet.
d
When a low yield is expected from the tissue, glycogen can be added prior to precipitation.
e
The pellet might not be visible and so mark the outside edge of the tube facing outwards in
the centrifuge, or ensure that the hinge of the lid is facing outwards during centrifugation.
f
DNA size and quality can be assessed by electrophoresis through a 1.5 % agarose gel and by
multiplex PCR (see Protocol 3.5) or a similar method as described by van Beers [40].
PROTOCOL 3.2 Column-Based DNA Isolation
from FFPE Tissue
g
Equipment and reagents
NucleoSpin Tissue XS Genomic DNA Purification from Tissue kit (Machery-Nagel)
•
Xylene
•
Ethanol [100% (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.
•
