Biomedical Engineering Reference
In-Depth Information
21 Add 2.5
×
the total volume of 100% (v/v) ethanol (ice cold).
b
22 Centrifuge at 12 000-16 000
g
for 15 min at room temperature.
23 Discard the supernatant and add 500
μ
l of 70% (v/v) ethanol (ice cold). Vortex the
sample and centrifuge at 20 000
g
for 10-15min at 4
◦
C.
24 Discard the supernatant and allow the pellet to air dry until no ethanol is visible.
25 Resuspend the pellet in 100
μ
lofTEorwater.
Notes
a
Do not vortex.
b
After mixing, the DNA should come out of solution.
PROTOCOL 1.2 DNA Extraction from FFPE Tissue
Equipment and reagents
•
Xylene (e.g. Merck - VEL, 90380)
•
Methanol
•
Ethanol (100% (v/v), 96% (v/v), 70%(v/v))
•
QIAamp DNA Mini Kit 250 (Qiagen, 51306), or QIAamp DNA Micro Kit 50 (Qiagen, 56304)
if the amount of tissue is limited (i.e. a biopsy)
•
NaSCN (e.g. Sigma), 1
M
Proteinase K (e.g. Roche), 20mg/ml
•
RNase A (e.g. Roche), 100mg/ml
•
Phosphate-buffered saline (PBS).
•
Method
c
1 Transfer two or three 50
μ
m FFPE tissue sections into a microcentrifuge tube.
2 Incubate with 1 ml of xylene for 7 min at room temperature, mixing a few times by
vortexing.
3 Centrifuge at 14 000
g
for 5 min at room temperature and discard the supernatant.
4 Repeat steps 2 and 3 twice.
5 Incubate with 1 ml of methanol for 5 min at room temperature, mixing a few times by
vortexing.
6 Centrifuge at 14 000
g
for 5 min at room temperature and discard the supernatant.
7 Repeat steps 5 and 6 once
