Bio-Rad SingleShot™ Cell Lysis RT-qPCR Kits - Manual (Page 5)

© 2014 Bio-Rad Laboratories, Inc. 10042473
SingleShot ™ Probes Kit
3. 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.
4. Add cDNA (prepared in the Preparation of Reverse Transcription Reactions section)
to the PCR tubes or wells containing qPCR reaction mix (prepared using Table 4),
seal tubes or wells with flat caps or optically transparent film, and vortex 30 sec
or more 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.
5. Program the thermal cycling protocol on a real-time PCR instrument according
to Table 5.
Table 5. Thermal cycling protocol.
* Shorter annealing/extension times (1–10 sec) can be used for amplicons <100 bp. Longer annealing/
extension times (30–60 sec or more) can be used for amplicons >250 bp, GC- or AT- rich targets,
low-expressing targets, crude samples, or higher input amounts (for example, 4 µl of cDNA).
Amplification
Real-Time PCR System
Setting/
Scan Mode
Polymerase
Activation
and DNA
Denaturation
Denaturation
at 95°C, sec
Annealing/
Extension
and Plate
Read at
60°C, sec* Cycles
Bio-Rad ® CFX96 ™ , CFX384 ™ ,
CFX96 Touch ™ , CFX96 Touch
Deep Well, CFX384 Touch ™ ,
CFX Connect ™
All channels
30 sec at
95°C for cDNA
5–15
10–30
35–40
Bio-Rad ® iQ ™ 5, MiniOpticon ™ ,
Chromo4 ™ , MyiQ ™
Standard 15–30
Applied Biosystems 7500
and 7900 HT, QuantStudio,
StepOne, StepOnePlus, ViiA 7 Fast 10–30
Standard 60
Roche LightCycler 96 or 480
Fast 10–30
Standard 60
QIAGEN Rotor-Gene and
Stratagene Mx series
Fast 10–30