Biomedical Engineering Reference
In-Depth Information
8 Add 1 ml of PBS, mixing a few times by vortexing.
9 Centrifuge at 14 000
g
for 5min at room temperature and discard the supernatant.
10 Repeat steps 8 and 9 once.
11 Incubate with 1 ml of 1
M
NaSCN overnight at 38-40
◦
C, mixing a few times by
vortexing.
12 Centrifuge at 14 000
g
for 5min at room temperature and discard the supernatant.
13 Wash the pellet three times with 1 ml of PBS as in steps 11 and 12.
14 Add 200
μ
l of Buffer ATL (QIAamp kit) and 20
μ
l of proteinase K, mixing a few times by
vortexing.
15 Incubate at 50-60
◦
C for 60 h, adding an extra 20
μ
l of proteinase K every 12 h.
16 Incubate with 40
μ
l of RNase A for 2 min at room temperature, mixing a few times by
vortexing.
17 Incubate with 400
μ
lofBufferAL(QIAampkit)for10minat65-75
◦
C, mixing a few
times by vortexing.
18 Add 420
μ
l of 100% (v/v) ethanol and mix by vortexing thoroughly.
19 Transfer 600
μ
l of the solution to a QIAamp centrifuge column.
20 Centrifuge at 2000
g
for 1 min at room temperature and discard the flow through.
21 Repeat steps 19 and 20 until all the sample has been applied to the column.
22 Add 500
μ
l of Buffer AW1 (QIAamp kit) to the column.
23 Centrifuge at 2000
g
for 1 min at room temperature and discard the flow through.
24 Add 500
μ
l of Buffer AW2 (QIAamp kit) to the column.
25 Centrifuge at 14 000
g
for 3 min at room temperature and discard the flow through.
26 Transfer the column to a fresh microcentrifuge tube (with a lid).
27 Elute the DNA from the column by adding 75
μ
l of Buffer AE (QIAamp kit), preheated to
65-75
◦
C.
28 Leave at room temperature for 1 min.
29 Centrifuge at 2000
g
for 1 min at room temperature.
30 Discard the column and store the DNA at 2-8
◦
C.
Notes
c
The protocol described is for the isolation of DNA from about 1 cm
2
or larger size tissue sections
using the QIAmp Mini kit. In the case of small-sized tissue sections (i.e. less than 0.5 cm
2
),
extract DNA using the QIAmp Micro kit. The proteinase K volumes and incubation times may
need to be adjusted, and the RNase treatment omitted. The quality of the DNA extracted from
FFPE tissue may differ considerably. In general, older paraffin blocks yield DNA of worse quality.
An important factor for preservation of DNA in FFPE tissue is the use of pH 7.0 buffered formalin
fixative before embedding in paraffin wax.
