Biomedical Engineering Reference
In-Depth Information
Fig. 1. Polymerase chain reaction
(
PCR) amplification for the
small sub-unit rRNA
(
ssrRNA
) gene of
Plasmodium falciparum
. Amplification bands of nested PCR visual-
ized on 1% agarose gel stained with ethidium bromide. Lanes 1 and 2, DNA extracted
from the plasma of malaria patients; lanes 3 and 4,: DNA extracted from the plasma of
healthy individuals; lane 5: no template control.
DNA. This control DNA is obtained from the extraction of DNA from erythrocyte
culture, which has been infected with the ring form of
P. falciparum.
The DNA
extracted should be quantified by a spectrophotometer (the mean of two readings
of two dilutions). From the DNA source, 10-fold serial dilutions should be made
ranging from 100,000 to 1 parasite genome equivalent per microliter (using the
conversion factor of 1 parasite genome equivalent = 0.02 pg). Make enough vol-
ume of each dilution for the experiments, aliquot, and freeze at -20°C.
2.
The TaqMan assay: the assay is specific for the
P. falciparum ssrRNA
gene. Primer
and probe sequences are as follows (GenBank accession no. M19173):
a. Fal-792F: 5'-GCT TTT GAG AGG TTT TGT TAC TTT GAG-3'.
b. Fal-889R: 5'-TTC CAT GCT GTA GTA TTC AAA CAC AAT-3'.
c. Fal-820T: 5'-(FAM) CTC AAT CAT GAC TAC CCG TCT GTT ATG AAC
ACT TAA TTT T (TAMARA)-3'.
Prepare dilutions of 5 pmols/
µ
L for the primers and probes with nuclease-free water.
3.4.2. Real-Time PCR Experiment
Analyzing the samples and standards in triplicate is recommended, and a
nontemplate control should be included in each run.
1.
Open the Sequence Detection System software and create a new file for your
experiment putting the type, name, and quantity (for standards) to each of the
96 wells in use (
see
Note 4
).
2.
Thaw all reagents and put on ice.
3.
Prepare a master mix from the following reagents (make 10% extra):
Nuclease-free water
5
µ
L per reaction
Taqman Universal Master Mix
12.5
µ
L per reaction
Forward primer
1.5
µ
L per reaction
Reverse primer
1.5
µ
L per reaction
Probe
0.5
µ
L per reaction
Total
21
µ
L per reaction
