Biomedical Engineering Reference
In-Depth Information
6.
0.5
M
EDTA, 0.5
M
Tris-HCl, pH 8.5.
7.
Wizard Resin Suspension and minicolumns (Promega, Madison, WI).
8.
5-mL Disposable syringes.
9.
Disposable tubes (1.5 mL and 50 mL).
10.
6
M
Guanidine isothiocyanate (GITC): 30 m
M
Tris-HCl, pH 7.0 (6
M
GITC).
11.
Column wash solution (CWS): 150 m
M
NaCl, 1 m
M
EDTA, 10 m
M
Tris-HCl,
pH 7.5, 60% ethanol.
12.
Nuclease-free water or TE (10 m
M
Tris-HCl, pH 8.0, 1 m
M
EDTA) for DNA
elution.
3. Methods
3.1. Urine Collection
All urine specimen preparation steps for Tr-DNA analysis are carried out at
room temperature unless otherwise stated. Routinely, 50-60 mL of urine were
collected for each specimen (
see
Note 1
). Immediately, but not later than
30 min after collection, 0.5
M
EDTA-0.5
M
Tris-HCl, pH 8.5 was added to
reach a final concentration of 10 m
M
(
see
Note 2
). Urine specimens were stored
in 5-mL aliquots at -20°C or for long-term storage at -80°C. In some instances,
cell-depleted urine was prepared before freezing by centrifugation for 10 min
at 800-1000
g
(
see
Note 3
).
3.2. DNA Isolation
1.
All steps of DNA purification are carried out at room temperature. 20 mL of 6
M
GITC was added to 10 mL of urine in a 50-cc tube and mixed vigorously. Usu-
ally, this step will solubilize particulate material that may be present in normal
urine specimens.
2.
For DNA adsorption, 0.2-0.25 mL Wizard Resin Suspension was then added and
the tubes placed on a rotating shaker for at least 2 h (
see
Note 4
). This incubation
step can be extended without significantly changing in DNA yield—we routinely
carried out this step overnight.
3.
The resin was pelletted by centrifugation at room temperature for 10 min at
2000
g
, after which the supernatant was carefully aspirated and discarded. The
resin pellet was resuspended in 1.0 mL of 4
M
GITC, and transferred into a 1.5-mL
microcentrifuge tube. This was then centrifuged in a benchtop microcentrifuge at
the top speed for 30-60 s. Again, the supernantant was removed and discarded.
4.
The pellet was resuspended in 1.0 mL of washing buffer and transferred in sus-
pension to a Promega Wizard minicolumn according to the recommendations of
the manufacturer. The column was then washed with an additional 2 mL of wash-
ing buffer. To remove residual washing buffer from the columns, they were cen-
trifuged for 2-3 min in a microcentrifuge at the maximum speed.
5.
The DNA was then eluted from each column with 50
L of water or TE. In order
to facilitate elution, each column-tube assembly can be incubated for 3-5 min in
µ
