Applied Biosystems 7300 Getting Started Guide

Appendix B Example RQ Experiment
84 Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
Notes
CHEMICAL HAZARD.
TaqMan Universal PCR Master Mix (2✕) No
AmpErase UNG 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.
v
5. Prepare the reaction plates.
a. Label the reaction plates, ensuring that you
include an endogenous control on each
plate.
b. Pipette 50 µL (20 µL for the Fast system) of
the appropriate PCR master mix (containing
cDNA) into each well of the plate.
c. Keep the reaction plates on ice until you are
ready to load them into the
7300/7500/7500 Fast system.
PCR Master Mix - Fast
Reaction
Component
µL/
Sample
µL/ 5
Reactions
‡
‡ 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.
Final
Concen-
tration
TaqMan Fast
Universal PCR
Master Mix (2✕)
10.0 50.0 1✕
20✕ TaqM a n
®
Gene Expression
Assay Mix
1.0 5.0 1✕
cDNA sample
and
Nuclease-free
water
9 µL 45 µL
10 to 100
ng
—
Total 20.0 100 —
GR2323
Regulus
Liver_96Plate.eps
GR2323
Endogenous
controls (GAPDH)
Liver
samples
GR2324
Regulus
Kidney_96Plate.eps
GR2324
Endogenous
controls (GAPDH)
Kidney
samples
GR2325
Regulus
Bladder_96Plate.eps
GR2325
Endogenous
controls (GAPDH)
Bladder
samples
Fast system plates are
notched in the A1
position on the top left
and have a
recommended 30 uL
volume capacity.

Applied Biosystems 7300 Specifications

General

Brand	Applied Biosystems
Model	7300
Category	Laboratory Equipment
Language	English