Biomedical Engineering Reference
In-Depth Information
c. Spin columns.
d. Collection tubes.
e. Wash buffer 1.
f. Wash buffer 2.
3.
Deionized water (dH
2
O).
2.3. Real-Time PCR Assay
1.
TaqMan PCR Reagent Kit (Perkin-Elmer):
a. Ampli
Taq
Gold polymerase (5 U/mL).
b. MgCl
2
(25 m
M
).
c. dCTP, dATP, dGTP (200
µ
M
each).
d. dUTP (400
M
).
e. Uracil-
N
-glycosylase (UNG) (1 U/
µ
µ
L).
f. 10X TaqMan Buffer A.
g. Forward primer, reverse primers and minor groove-binding (MGB) probe
(Applied Biosystems) (
Table 1
).
h. Molecular-grade dH
2
O.
i. Dimethylsulfoxide (DMSO) 100%.
2.
Namalwa cell line (American Type Culture Collection no. CRL-1432).
3.
Applied Biosystems ABI PRISM 7700 sequence detector.
4.
Applied Biosystems MicroAmp Optical 96-well reaction plates.
5.
Applied Biosystems optical caps (8 caps/strip).
3. Methods
3.1. Sample Types and Preparation
1.
Collect peripheral blood into a tube containing EDTA as anti-coagulant.
2.
Centrifuge the sample at 1600
g
for 10 min to separate plasma from cellular com-
ponents.
3.
Carefully transfer the supernatant into a 1.5-mL polypropylene tube without dis-
turbing the cellular components.
4.
Microcentrifuge the polypropylene tube at 16,000
g
for 10 min to ensure the
removal of all cells.
5.
Transfer the supernatant (cell-free plasma) into a clean polypropylene tube for
subsequent DNA extraction (
see
Note 1
).
6.
Plasma samples used for size analysis of circulating DNA should be processed
within 6 h of blood collection (
see
Note 2
).
7.
If the samples are to be used in different analyses carried out at different times,
the plasma samples should be aliquoted into smaller fractions to avoid repeated
freezing and thawing, or DNA should be extracted from the plasma samples for
storage (
see
Note 2
).
