Biomedical Engineering Reference
In-Depth Information
2. Materials
2.1. Equipment
1.
Centrifuge.
2.
Micro-centrifuge.
3.
Vortex.
4.
Water bath.
5.
PCR machine.
6.
Agarose gel electrophoresis equipment.
7.
Sequence Detection System (real-time PCR).
8.
Pipets for PCR.
9.
Filter tips.
2.2. Reagents
1.
QIAamp DNA Blood Mini Kit (Qiagen).
2.
PCR kit.
3.
Primers and probes.
4.
TaqMan Universal Master Mix (Applied Biosystems).
5.
MicroAmp Optical 96-well Reaction Plate (Applied Biosystems).
6.
MicroAmp Adhesive Covers or Optical Caps (Applied Biosystems).
7.
Nuclease-free water.
3. Methods
The methods described in
Subheadings 3.1.-3.4.
outline (1) blood collec-
tion and plasma separation, (2) extraction of DNA from plasma, (3) detection
of
P. falciparum
parasite using a nested PCR assay, and (4) quantitation of
P.
falciparum
DNA in plasma by real-time PCR.
3.1. Blood Collection and Plasma Separation
1.
Collect blood into ethylenediamine tetraacetic acid (EDTA) tubes. Keep blood
samples at room temperature and process the blood as soon as possible.
2.
Centrifuge samples at 1000
g
for 10 min at room temperature.
3.
Transfer the plasma layer into a clean, labeled, 15-mL falcon tube. Take extra
care to not disturb the buffy coat layer.
4.
Spin the plasma again (as in
step 2
).
5.
Remove the plasma without disturbing the pellet and transfer it into clean,
labeled, DNase-free tubes and store at -70°C.
3.2. Extraction of DNA From Plasma
DNA is extracted from plasma using the QIAamp Blood Mini Kit (Qiagen).
The protocol is described as follows, but for more details and troubleshooting,
please refer to the kit handbook.
