Biomedical Engineering Reference
In-Depth Information
Method
1 To a 1.5 ml microcentrifuge tube add:
•
0.5-1cm
3
of tissue;
•
600
μ
l of EDTA/nuclei lysis solution;
•
17.5
μ
lofproteinaseK.
2 Incubate overnight at 55
◦
C with gentle shaking, or vortex the sample several times
during the incubation.
3Add3
μ
l of RNase solution to the nuclear lysate and mix the sample by inverting the
tube two to five times.
4 Incubate the mixture for 15-30min at 37
◦
C.
5 Add 200
μ
l of protein precipitation solution to the sample and vortex vigorously. Chill
thesampleonicefor10min.
6 Centrifuge at 20 000
g
for 15min at room temperature to pellet the precipitated
protein.
7 Carefully transfer the supernatant containing the DNA to a fresh 1.5 ml microcentrifuge
tube.
8 Add 600
μ
l of isopropanol (at room temperature).
9 Mix the solution by gently inverting until the white thread-like strands of DNA form a
visible mass.
10 Centrifuge at 20 000
g
for 1min at room temperature. The DNA will be visible as a small
white pellet. Carefully aspirate supernatant by decanting the liquid. Air dry until no
ethanol is visible.
11 Add 200
μ
l of TE to resuspend the DNA.
12 Pellet 2ml of PLG light by centrifuging at 12 000-16 000
g
for 20-30s at room
temperature.
13 Add the 200
μ
l of DNA-containing TE to the 2 ml PLG light tube, followed by 200
μ
lof
phenol-chloroform.
14 Mix the organic and the aqueous phases thoroughly by inversion.
a
15 Centrifuge at 12 000-16 000
g
for 5min at room temperature to separate the phases.
Transfer the upper layer/supernatant to a new 1.5 ml microcentrifuge tube.
16 Add 200
μ
l of chloroform directly to the above new 1.5 ml microcentrifuge tube.
17 Mix thoroughly by inversion.
a
18 Centrifuge at 12 000-16 000
g
for 5min at room temperature to separate the
phases.
19 Transfer the aqueous solution (above the gel) to a new 1.5ml microcentrifuge
tube.
20 Add 20
μ
lof3
M
sodium acetate and mix by inversion.
