Bio-Rad SingleShot™ Cell Lysis RT-qPCR Kits User Manual
Page 7
© 2014 Bio-Rad Laboratories, Inc.
10042473
SingleShot
™
Probes Kit
4. Perform the reverse transcription reactions following the previously
mentioned protocol.
5. Prepare the qPCR reactions following the recommendations in Table 7. Do not add
cDNA until step 8. Maintain the same input lysate, cDNA and qPCR volumes for all
reactions in this experiment.
If desired, gene expression targets of interest with non-HEX probes can
be amplified in parallel with the RNA control assay. Adjust volumes in
Table 7 accordingly
6. Mix the qPCR reaction mix thoroughly to ensure homogeneity and dispense equal
aliquots into each PCR tube or into the wells of a PCR plate. Use good pipetting
technique to ensure assay precision and accuracy.
7. Add cDNA to the PCR tubes or wells containing qPCR reaction mix (prepared
using Table 7), seal tubes or wells with flat caps or optically transparent film, and
gently vortex to ensure thorough mixing of the reaction components. Spin the
tubes or plate to remove any air bubbles and to collect the reaction mixture in
the vessel bottom.
8. Program the thermal cycling protocol on a real-time PCR instrument according
to Table 5.
* Scale all components proportionally according to sample number and reaction volumes.
Table 7. Preparation of qPCR reaction mix for RNA control assay.*
Component
Volume per 10 µl
Reaction, µl
Volume per 20 µl
Reactions, µl
Final
Concentration
SsoAdvanced Universal Probes
Supermix (2x)
5
10
1x
SingleShot
™
Probes qPCR
Control Assay
0.5
1
1x
cDNA (add at step 8)
1–2
2–4
—
Nuclease-Free water
Variable
Variable
—
Total reaction mix volume
10
20
—