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

© 2014 Bio-Rad Laboratories, Inc. 10042474
SingleShot ™ SYBR ® Green Kit
* Scale all components proportionally according to sample number and reaction volumes.
Table 4. qPCR reaction setup.*
Component
Volume per 10 µl
Reaction, µl
Volume per 20 µl
Reactions, µl
Final
Concentration
SsoAdvanced ™ Universal SYBR ®
Green Supermix (2x)
5 10 1x
Forward and Reverse Primers Variable Variable 250–500 nM each primer
cDNA (add at step 4) 1–2 2–4 —
Nuclease-Free Water Variable Variable —
Total reaction mix volume 10 20 —
Preparation of qPCR Reactions
Instrument Compatibility
SsoAdvanced ™ Universal SYBR ® Green 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 SYBR ® Green Supermix and other frozen reaction
components to room temperature. Mix thoroughly, centrifuge briefly to collect
solutions 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.
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 according to
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.