Download Relative Quantification Getting Started Guide for the Applied

Chapter 1 Introduction and Example RQ Experiment
Example RQ Experiment
c. Program the thermal cycler using the
indicated parameter values for the RT step
of the two-step RT-PCR method.
Step Type
Time
Temperature
HOLD
10 min
25 °C
Note: You have the option to use one-step
HOLD
120 min
37 °C
RT-PCR, as explained in “Selecting One- or
Two-Step RT-PCR” on page 20.
d. Store the cDNA at −20 °C until use.
4. Prepare the PCR master mix as indicated in the
PCR Master Mix - Standard
table to the right.
See Chapter 4 on page 28 for more information.
Note: The reaction volumes for TaqMan® Gene
Expression Assays and TaqMan® Custom Gene
Expression Assays are specified in the product
insert. Those for primers and probes designed
with Primer Express software follow the
universal assay conditions described in
Chapter 4.
CHEMICAL HAZARD.
TaqMan Universal PCR Master Mix may cause eye
and skin irritation. Exposure may cause discomfort if
swallowed or inhaled. Read the MSDS, and follow
the handling instructions. Wear appropriate protective
eyewear, clothing, and gloves.
CHEMICAL HAZARD.
TaqMan Universal PCR Master Mix may cause eye
and skin irritation. Exposure may cause discomfort if
swallowed or inhaled. Read the MSDS, and follow
the handling instructions. Wear appropriate protective
eyewear, clothing, and gloves.
Reaction
Component
µL/
Sample
µL/ 5
Reactions b
Final
Concentration
TaqMan Universal
PCR Master Mix
(2✕)
25.0
125.0
1✕
20✕ TaqMan®
Gene Expression
Assay Mix a
2.5
12.5
1✕
cDNA sample
5.0
25.0
10 to
100 ng
Nuclease-free
water
17.5
87.5
—
Total
50.0
250
—
a. Contains forward and reverse primers and labeled probe.
b. 24 master mixes are prepared, one for each of 23 genes plus the
endogenous control. Volume for five reactions (4 replicates plus extra)
to account for pipetting losses.
PCR Master Mix - Fast
µL/
Sample
µL/ 5
Reactionsa
Final
Concentration
TaqMan Fast
Universal PCR
Master Mix (2✕)
10.0
50.0
1✕
20✕ TaqMan®
Gene Expression
Assay Mix
1.0
5.0
1✕
Reaction
Component
cDNA sample
and
9 µL
45 µL
Nuclease-free
water
Total
10 to 100
ng
—
20.0
100
—
Notes
8
Relative Quantification Getting Started Guide for the Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System