Biomedical Engineering Reference
In-Depth Information
Concentration
5X Generic reagents
in final reaction
250 m
M
Tris, pH 8.3
50 m
M
2.5 mg/mL BSA (
see
Note 1
)
500
µ
g/mL
1 m
M
of each dNTP (dATP, dCTP, dGTP, and dTTP)
200
µ
M
each
15 m
M
MgCl
2
(
see
Note 2
)
3 m
M
2 U/10
µ
L heat-stable polymerase (
see
Note 3
)
0.4 U/10
µ
L
5X dye (
see
Note 4
)
1X
2.2. Oligonucleotide Mixture
This mixture includes all primers and probes. For a 5X mixture, the primers
are at 2.5
µ
M
and probes at 1.0
µ
M
, resulting in primer and probe concentra-
tions of 0.5
µ
M
, respectively, in the reaction (
see
Note 5
). The
oligonucleotide mixture is prepared in 1X TE (10 m
M
Tris, 0.1 m
M
ethylene-
diamine tetraacetic acid [EDTA], pH 8.0) and is stable for weeks at 4°C and
months at -20°C.
µ
M
and 0.2
2.3. Template Solution
The typical amount of template per 10-
10
4
cop-
ies. This is approx 50 ng mammalian DNA (
see
Note 6
), 50 pg bacterial DNA,
0.17 pg viral DNA, or 0.01 pg plasmid DNA. Template (in 1X TE') is usually
diluted 2- to 10-fold in the final PCR solution. A fivefold dilution is assumed
in the following. If absolute standards are used, it is best to dilute them from a
concentrated stock solution (10
5
-10
10
copies per
µ
L reaction is approx 1.6
×
L) just before use. Some
templates (particularly diluted PCR products) are not stable for more than 24 h
at 4-25°C. If RNA templates are of interest, reverse transcription is necessary
(
see
Note 7
).
µ
3. Methods
3.1. Primer Design
Primer design for real-time PCR is similar to primer design for regular PCR.
The following guidelines are useful:
1.
Use primers between 15 and 30 bases that are matched in melting temperature
(T
m
) to each other. A primer greater than 17 bases long has a good chance of being
unique in the human genome.
2.
Unless there is a reason to amplify longer targets, choose a product length less
than 500 bp, preferably less than 200 bp. Shorter products amplify with higher
efficiency.
3.
Avoid primers that anneal to themselves or to other primers, particularly at
their 3' ends.
