Bio-Rad SingleShot™ Cell Lysis RT-qPCR Kits User Manual
Page 4
© 2014 Bio-Rad Laboratories, Inc.
10042473
SingleShot
™
Probes Kit
2. Incubate the complete reaction mix in a thermal cycler using the following protocol:
reverse transcription for 30 min at 42°C followed by RT inactivation for 5 min
at 85°C.
3. Proceed to the Preparation of qPCR Reactions section or store the cDNA
at –20°C.
Recommendations for the use of no-RT control
■
■
Interference of gene expression analysis by gDNA carryover in cell lysate
samples can be tested by setting up a no-RT control reaction
■
■
The reverse transcriptase volume in a no-RT control reaction should be replaced
with water
■
■
The same amount of cell lysate used in the RT reaction should be used in the
no-RT reaction to ensure similar carryover of cDNA synthesis components in a
qPCR reaction
Preparation of qPCR Reactions
Instrument Compatibility
SsoAdvanced Universal Probes Supermix is compatible with all Bio-Rad and other
commercially available real-time PCR systems.
Reaction Mix Preparation and Thermal Cycling Protocol
1. Thaw SsoAdvanced Universal Probes Supermix and other frozen reaction
components to room temperature. Mix thoroughly, centrifuge briefly to collect
solution at the bottom of tubes, then store on ice protected from light.
2. Prepare (on ice or at room temperature) enough qPCR reaction mix for all qPCR
reactions by adding all required components, except the template, according to
the recommendations in Table 4.
Table 4. qPCR reaction setup.*
* Scale all components proportionally according to sample number and reaction volumes.
** For duplex assays with large ΔCq (ΔC
T
) values, decreasing the primer concentrations for the higher-
expressing target may help. To validate, perform a primer matrix to determine optimal final primer
concentration. Cq, quantification cycle; C
T
, threshold cycle.
Component
Volume per 10 μl
Reaction, µl
Volume per 20
Reactions, µl
Final Concentration
SsoAdvanced Universal
Probes Supermix (2x)
5
10
1x
Forward and
Reverse Primers
Variable
Variable
250–900 nM**
each primer
Fluorogenic Probe
Variable
Variable
150–250 nM each probe
cDNA (add at step 4)
1–2
2–4
—
Nuclease-Free Water
Variable
Variable
—
Total reaction
mix volume
10
20
—